Drugs, foods or drinks with the use of algae-derived physiologically active substances

ABSTRACT

Medicinal compositions for treating, ameliorating or preventing diseases with sensitivity to 3,6-anhydrogalactopyranose represented by formula (1):  
                 
foods, drinks, cosmetics, etc. containing as the active ingredient at least one member selected from the group consisting of the above-mentioned compound, its aldehyde, its hydrate and 2-O-methylated derivatives thereof and soluble sugar compounds containing the above compound. This compound also shows, for example, an apoptosis-inducing activity, a carcinostatic activity and inhibitory activities on the production of active oxygen, lipid peroxide radicals and NO, which makes it useful also as the active ingredient of antioxidants and preservatives.

This is a division of parent application Ser. No. 10/228,195 filed Aug.27, 2002, itself a division of grandparent application Ser. No.09/554,235, now U.S. Pat. No. 6,475,990, issued on Nov. 5, 2002.

FIELD OF THE INVENTION

The present invention relates to use of a physiologically activesubstance derived from algae. More specifically, it relates to apharmaceutical composition, an antioxidant, a preservative compositionfor keeping freshness of foods and drinks, and a cosmetic compositionwhich comprise the physiologically active substance as an activeingredient, as well as a functional food or drink which comprises thephysiologically active substance. Furthermore, it relates to asaccharide for exhibiting the function.

BACKGROUND OF THE INVENTION

Recently, a mode of death of cells or tissues called as apoptosis(self-blasting or self-destruction of cells) has been noticed.

The apoptosis is a death which has been originally programmed in thegenome of a cell and is different from necrosis which is a pathologicalcell death. Certain external or internal factors trigger the activationof a gene that programs the apoptosis to cause the biosynthesis of aprogrammed death protein. In some cases, a programmed death proteinwhich has been present in a cell in its inactive form becomes activated.The active programmed death protein thus formed decomposes the cell tolead death.

Activation of the apoptosis in desired tissues or cells would make itpossible to eliminate cells which are unnecessary or harmful from aliving body in a natural manner, which is of very importance.

OBJECTS OF THE INVENTION

Oligosaccharides derived from algae such as agar are expected to bedeveloped as raw materials for foods (Food Chemical, 1988-2, 40-44;Bessatsu Food Chemical (Extra Number Food Chemical)-4, 1990, December,127-131; JP-A-6-38691). However, their physiological functions such asan apoptosis-inducing activity are unknown.

The main object of the present invention is to develop a highly safesubstance having a physiological function such as an activity ofinducing apoptosis derived from a naturally occurring material, as wellas to provide a pharmaceutical composition for preventing or treating adisease sensitive to the substance, such as a composition for inducingapoptosis comprising the substance as an active ingredient, and afunctional food or drink comprising the substance as a constituentcomponent.

SUMMARY OF THE INVENTION

In brief, the first aspect of the present invention is a pharmaceuticalcomposition which comprises as an active ingredient at least one memberselected from the group consisting of:

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1:

an aldehyde and a hydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound at its reducing end, saidcomposition being used for treating or preventing a disease sensitive tothe compound.

The second aspect of the present invention is a food or drink comprisingat least one member selected from the group consisting of:

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1, an aldehyde and ahydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound at its reducing end,

said food or drink being used for ameliorating a disease state of orpreventing a disease sensitive to the compound.

The third aspect of the present invention is an antioxidant whichcomprises as an active ingredient at least one member selected from thegroup consisting of

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1, an aldehyde and ahydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound.

The fourth aspect of the present invention is a food and drinkcomprising the antioxidant of the third aspect of the present invention.

The fifth aspect of the present invention is a saccharide for anantioxidant selected from the group consisting of:

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1, an aldehyde and ahydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound.

The sixth aspect of the present invention is a preservative compositionfor keeping freshness of foods and drinks which comprises as an activeingredient at least one member selected from the group consisting of:

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1, an aldehyde and ahydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound.

The seventh aspect of the present invention is a cosmetic compositioncomprising as an active ingredient at least one saccharide selected fromthe group consisting of agarobiose, agarotetraose, agarohexaose,agarooctaose, K-carabiose andβ-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose.

The eighth aspect of the present invention is an acidic food or drinkcomprising at least one member selected from the group consisting of:

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1, an aldehyde and ahydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound.

The further aspect of the present invention is use of at least onemember selected from the group consisting of:

a compound selected from the group consisting of3,6-anhydrogalactopyranose represented by formula 1, an aldehyde and ahydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and

a soluble saccharide containing the compound, in preparation of apharmaceutical composition, a food or drink, an antioxidant, apreservative composition for keeping freshness of foods and drinks or acosmetic composition.

Hereinafter, the present invention will be explained in detail withreference to the attached drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a gel filtration elution pattern of agar decomposedwith 0.12 N HCl.

FIG. 2 illustrates a gel filtration elution pattern of agar decomposedwith 1 N HCl.

FIG. 3 illustrates a size-exclusion HPLC chromatogram of agar decomposedwith an acid.

FIG. 4 illustrates a mass spectrum of an apoptosis-inducing andcarcinostatic substance.

FIG. 5 illustrates a ¹H-NMR spectrum of an apoptosis-inducing andcarcinostatic substance (hydrate form).

FIG. 6 illustrates a ¹H-NMR spectrum of an apoptosis-inducing andcarcinostatic substance (aldehyde form).

FIG. 7 illustrates an elution pattern of normal phase HPLC ofagarobiose, agarotetraose and agarohexaose.

FIG. 8 illustrates a mass spectrum of the peak at 66.7 min.

FIG. 9 illustrates a mass spectrum of the peak at 78.5 min.

FIG. 10 illustrates a mass spectrum of the peak at 85.5 min.

FIG. 11 illustrates a mass spectrum of 3,6-anhydro-L-galactose.

FIG. 12 illustrates a ¹H-NMR spectrum of 3,6-anhydro-L-galactose(hydrate).

FIG. 13 illustrates a ¹H-NMR spectrum of 3,6-anhydro-L-galactose(aldehyde).

FIG. 14 illustrates the relation between the incubation time and thenumber of viable cells obtained by incubating HL-60 cells with additionof one of oligosaccharides at a final concentration of 250 μM.

FIG. 15 illustrates the relation between the incubation time and thenumber of viable cells obtained by incubating HL-60 cells with additionof one of oligosaccharides at a final concentration of 125 μM.

FIG. 16 illustrates an elution pattern in normal phase HPLC chromatogramof agar treated by heating in 0.5 M phosphate.

FIG. 17 illustrates a calibration curve of agarobiose.

FIG. 18 illustrates the relation between the heating time and the amountof agarobiose produced in 0.2% agar solution in 0.1 M HCl.

FIG. 19 illustrates the relation between the heating time and the amountof agarobiose produced in 0.2% agar solution in 0.1 M citric acid.

FIG. 20 illustrates the production of agaro-oligosaccharides in 500 mMcitric acid at 80° C.

FIG. 21 illustrates the production of agaro-oligosaccharides in 500 mMcitric acid at 95° C.

FIG. 22 illustrates the production of agaro-oligosaccharides in 1200 mMlactic acid at 80° C.

FIG. 23 illustrates the production of agaro-oligosaccharides in 1200 mMlactic acid at 95° C.

FIG. 24 illustrates the production of agaro-oligosaccharides in 1000 mMmalic acid at 80° C.

FIG. 25 illustrates the production of agaro-oligosaccharides in 1000 mMmalic acid at 95° C.

FIG. 26 illustrates the production of agaro-oligosaccharides in 1000 mMmalic acid at 70° C.

FIG. 27 illustrates a normal phase HPLC chromatogram of K-carrageenandecomposed with an acid.

FIG. 28 illustrates a mass spectrum of an apoptosis-inducing andcarcinostatic substance.

FIG. 29 illustrates a ¹H-NMR spectrum of an apoptosis-inducing andcarcinostatic substance.

FIG. 30 illustrates the results of gel filtration with CellulofineGCL-25.

FIG. 31 illustrates the results of gel filtration with a Sephadex LH-20column.

FIG. 32 illustrates a mass spectrum ofS-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose.

FIG. 33 illustrates a ¹H-NMR spectrum ofS-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose.

FIG. 34 illustrates the relation between the concentration of agarobioseand the level of ³H-thymidine uptake in lymphocyte blastgenesis inducedby ConA.

FIG. 35 illustrates the relation between the concentration of agarobioseand the level of ³H-thymidine uptake in a mixed lymphocyte reaction.

FIG. 36 illustrates NO₂ ⁻ concentrations in culture media in thepresence of various concentrations of agarobiose.

FIG. 37 illustrates NO₂ ⁻ concentrations in culture media in thepresence of various concentrations of neoagarobiose.

FIG. 38 illustrates NO₂ ⁻ concentrations in culture media in thepresence of a solution of agar digested by hydrochloric acid or citricacid.

FIG. 39 illustrates NO₂ ⁻ concentrations in culture media in thepresence of 3,6-anhydro-D-galactose or galactose.

FIG. 40 illustrates NO₂ ⁻ concentrations in culture media under variousconditions.

FIG. 41 illustrates carcinostatic activity of the oligosaccharide of thepresent invention.

FIG. 42 illustrates inhibition of PCA reaction by the oligosaccharide ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

An aldehyde of 3,6-anhydrogalactopyranose of formula 1 (hereinaftersimply referred to as “3,6-anhydro-galactopyranose”) of the presentinvention is a compound of formula 2:

A hydrate thereof is a compound of formula 3:

A 2-O-methylated derivative of the 3,6-anhydrogalactopyranose is acompound of formula 4:

An aldehyde of the methylated derivative is a compound of formula 5:

A hydrate of the methylated derivative is a compound of formula 6:

The structures of formulas 1 to 6 used herein may be represented bydifferent expression forms. It is intended that such differentexpression forms and their possible tautomers are included in formulas 1to 6. In addition, the configuration of formulas 1 to 6 is not limitedto specific one as far as the desired activities are exerted, and may bein the D-form or L-form, or a mixture thereof.

The soluble saccharide of the present invention is, without limitation,a soluble saccharide containing at least one compound selected from3,6-anhydrogalactopyranose, an aldehyde and a hydrate thereof, and2-O-methylated derivatives of the 3,6-anhydrogalactopyranose, thealdehyde and the hydrate (hereinafter collectively referred to as “thecompounds of formulas 1 to 6”), and can be obtained by decomposition ofa substance containing at least one compound selected from the compoundsof formulas 1 to 6 (hereinafter simply referred to as “a raw substance”)under acidic conditions below pH 7 with an acid and/or enzyme, or bychemical synthesis. The soluble saccharide of the present invention isnot limited to specific one in so far as it dose not solidify orsemi-solidify (gelate) when used. Therefore, any saccharides containingat least one compound selected from the compounds of formulas 1 to 6which become isolated when used are included in the soluble saccharidesof the present invention. Examples of the soluble saccharides suitablyused in the present invention include a saccharide whose non-reducingend is a sugar other than L-galactose-6-sulfate, for example,agarobiose, agarotetraose, agarohexaose, agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.

The raw substances used for obtaining the soluble saccharides are notlimited to specific one and include, for example, viscouspolysaccharides from red algae such as agarose, agaropectin, funoran,porphyran, carrageenan, furcellaran, and hypnean [Kyoritsu-shuppan Inc.,“Tatouseikagaku 1—Kagakuhen—(Biochemistry of Polysaccharides1—Chemistry—), pp. 314 (1969)].

The raw substances also include materials that contain thesepolysaccharides. For example, as raw materials for agarose andagaropectin, red algae belonging to Gelidiaceae such as Gelidiumamansii, Gelidium japonicum, Gelidium pacificum, Gelidium subcostatum,Pterocladia tenuis, Acanthopeltis japonica and the like, red algaebelonging to Gracilariaceae such as Gracilaria verrucosa, Gracilariagigas and the like, red algae belonging to Ceramiaceae such as Ceramiumkondoi, Campylaephora hypnaeoides and the like, and other red algae areused. Usually, several kinds of algae are used in combination as the rawmaterials. Although algae dried in the sun are usually used as the rawmaterials, both fresh and dried algae can be used in the presentinvention. Algae which are bleached while spraying water during drying,i.e., bleached raw algae, can also be used.

The raw material algae are extracted with hot water and then cooled toobtain “gelidium jelly”. Water is removed from this “gelidium jelly” byfreeze-dehydration or compress-dehydration, followed by drying to obtainagar. Agar in various forms such as bar, belt, board, thread, powder andthe like can be used regardless of the source algae. Usually, agarcontains about 70% of agarose and about 30% of agaropectin. The agar canbe further purified to prepare agarose with high purity. Purifiedagarose with high purity or law purity having various agarose contentscan be used.

The raw substances include the above-mentioned raw material algae,gelidium jelly, agar, purified agarose, purified agaropectin andintermediate products or side products obtained during preparation ofthese substances.

Agarose is a polysaccharide whose main structure is alternately linkedD-galactose and 3,6-anhydro-L-galactose. In the structure, 1-position ofD-galactose and 4-position of 3,6-anhydro-L-galactose are linked to eachother through S-glycoside bond and 1-position of 3,6-anhydro-L-galactoseand 3-position of D-galactose are linked to each other throughα-glycoside bond. The α-1,3-bond is hydrolyzed by mild hydrolysis with adilute acid or α-agarase [Carbohydr. Res., Vol. 66, p. 207 (1978)], andthe β-1,4-bond is hydrolyzed by β-agarase selectively.

Carrageenan is a polysaccharide which is contained in red algae such asGigartinaceae, Solieriaceae, Hypneaceae and the like. κ-Carrageenan,λ-carrageenan and η-carrageenan are known.

κ-Carrageenan has a fundamental structure in which 1-position ofD-galactose-4-sulfate is linked to 4-position of 3,6-anhydro-D-galactosethrough β-glycoside bond, 1-position of 3,6-anhydro-D-garactose islinked to 3-position of D-galactose-4-sulfate through α-glycoside bond,and they are repeated alternately. λ-Carrageenan has a fundamentalstructure in which 1-position of D-galactose is linked to 4-position ofD-galactose-2,6-disulfate through β-glycoside bond, 1-position ofD-galactose-2,6-disulfate is linked to 3-position of D-galactose throughα-glycoside bond, and they are repeated alternately. Carrageenan isutilized as a gelatinizing agent of foods.

The raw substances of the present invention also include partiallydecomposed products of the above-mentioned raw substances using achemical, physical and/or enzymatic method.

Examples of chemical decomposition include hydrolysis under acidic toneutral conditions. Examples of physical decomposition include radiationof electromagnetic waves or ultrasonic waves. Examples of enzymaticdigestion include hydrolysis with a hydrolase such as agarase,carrageenase and the like.

Decomposition of the raw substances under acidic to neutral conditionsare not limited to specific one in so far as the decomposition producesthe compounds of formulas 1 to 6 and the soluble saccharides containingat least one of these compounds which have an apoptosis-inducingactivity; a carcinostatic activity; antioxidant activities such as anactivity of inhibiting active oxygen production, an activity ofinhibiting nitrogen monoxide (hereinafter referred to as NO) production;an immunoregulatory activity; or the like. Examples of the saccharidesinclude agarobiose, agarotetraose, agarohexaose, agarooctaose,κ-carabiose (hereinafter simply referred to “carabiose”),β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose, and the like;and the saccharides containing the compounds selected from the compoundsof formulas 1 to 6 at their reducing ends whose non-reducing ends aresaccharides other than L-galactose-6-sulfate.

For example, the raw substance is dissolved or suspended in an acid andreacted to produce the compound selected from the compounds of formulas1 to 6 and the soluble saccharides containing at least one of thesecompounds to be used in the present invention. The reaction timerequired for the production of the compound selected from the compoundsof formulas 1 to 6 and the soluble saccharides containing at least oneof these compounds can be reduced by heating upon reaction.

The kind of the acid to be used for dissolution or suspension of the rawsubstances (for example, a substance that contains agarose or anagarose) is not limited to a specific one and may be inorganic acidssuch as hydrochloric acid, sulphuric acid, nitric acid and the like,organic acids such as citric acid, formic acid, acetic acid, lacticacid, ascorbic acid and the like, solid acids such as cation exchangeresins, cation exchange fibers, cation exchange membranes and the like.

The concentration of the acid is not limited, but the acid can be usedat a concentration of 0.0001 to 5 N, preferably 0.01 to 1 N. Inaddition, the reaction temperature is not limited, but the reaction maybe carried out at 0 to 200° C., preferably 20 to 130° C. Furthermore,the reaction time is not limited, but the reaction may be carried outfor a few seconds to a few days. The kind and the concentration of theacid, the reaction temperature and the reaction time may be suitableselected depending on the particular kind of the raw substancecontaining at least one compound selected from the compounds of formulas1 to 6, such as agarose or carrageenan, as well as the compound ofinterest selected from the compounds of formula 1 to 6, the yield of thesaccharide containing the compound, and the degree of polymerization ofthe soluble saccharide of interest containing the compound selected fromthe compounds of formulas 1 to 6 at its reducing end. In general, theacid decomposition reaction proceeds more rapidly by selecting a strongacid rather than a weak acid, a high acid concentration rather than alow acid concentration, and a high temperature rather than a lowtemperature.

Furthermore, in general, when a solid acid is used, a strong cationicexchange resin gives better decomposition reaction efficiency than aweak cationic exchange resin does. In addition, when the amount of thesolid acid relative to the amount of the raw substance is more and thereaction temperature is higher, the acid decomposition reaction proceedsmore rapidly.

For example, a solution of the saccharide used in the present inventionwhich is obtained by suspending agar in 0.1 N hydrochloric acid in anamount of 10% by weight, dissolving the agar by heating at 100° C. for13 minutes and removing insoluble materials does not gelate any longereven when the solution is cooled to its freezing point. When thesaccharide contained in this solution is analyzed by gel filtrationHPLC, normal phase HPLC and the like, saccharides with high molecularweight are scarcely observed and almost all of the saccharides aredecomposed to soluble saccharides composed of 10 or less sugars.Likewise, in case of a solid acid, a solution of the saccharide of thepresent invention obtained by converting 1 part by weight of a Na-typecommercially available strong cationic exchange resin to its H type with1 N hydrochloric acid, placed in 79 parts by weight of deionized water,adding and suspending 10 parts by weight of agar and heating the mixtureat 95° C. for 180 minutes dose not gelate any longer, even when thesolution is cooled to its freezing point. When the saccharide containedin this solution is analyzed by gel filtration HPLC, normal phase HPLCand the like, saccharides with high molecular weight are scarcelyobserved and almost all of the saccharides are decomposed to solublesaccharides composed of 10 or less sugars.

Furthermore, for producing the soluble saccharide used in the presentinvention which has the compound selected from the compounds of formulas1 to 6 at its reducing end, a large amount of the physiologically activeoligosaccharide, such as a saccharide for an antioxidant, can beproduced by using an organic acid such as citric acid, lactic acid ormalic acid, suitably selecting the acid concentration ranging fromseveral 10 mM to several M, the heating temperature ranging from 70 to95° C., and the heating time ranging from several 10 minutes to 24hours. In addition, the physiologically active oligosaccharide producedhas long-term storage stability if it is maintained under acidicconditions while preventing them from becoming alkaline afterhydrolysis.

The decomposed raw substances may be used directly or after beingneutralized as the compounds to be used in the present invention, i.e.,the compound selected from the compounds of formulas 1 to 6 and thesoluble saccharides containing at least one of these compounds, forexample, saccharides such as agarobiose, agarotetraose, agarohexaose,agarooctaose, κ-carabiose andS-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.However, they may be further purified. The compound selected from thecompounds of formulas 1 to 6 and the soluble saccharides containingthese compounds at their reducing ends, for example, an oligosaccharidesuch as agarobiose, agarotetraose, agarohexaose, agarooctaose,κ-carabiose and β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactosecan be purified by using, for example, its apoptosis-inducing activityor carcinostatic activity as an index. As the means for purification, aknown method such as a chemical method, a physical method or the likecan be used. The compound selected from the compounds of formulas 1 to 6or the soluble saccharide containing at least one of the compounds,which is an apoptosis-inducing substance, produced in the aciddecomposition products can be purified by combining known purificationmethods such as gel filtration, fractionation using a molecular weightfractionating membrane, solvent extraction and chromatography using ionexchange resins or the like.

The structures of the resultant compounds can be analyzed by the knownmethods such as mass spectrometry, nuclear magnetic resonance,measurement of ultraviolet absorption spectrum or infrared absorptionspectrum and the like.

Agarobiose, one example of the active ingredient of the presentinvention, is a disaccharide in which 1-position of D-galactose and4-position of 3,6-anhydro-L-galactose are linked to each other throughβ-glycoside bond. An α-isomer and a β-isomer exist because an anomercarbon is present at 1-position of 3,6-anhydro-L-galactose, andagarobioses to be used in the present invention include both of theisomers.

The saccharide containing the compound selected from the compounds offormulas 1 to 6 at its reducing end used as the active ingredient in thepresent invention is one in which one or more sugars are bound to one ormore hydroxide groups other than that at 1-position of the compoundselected from the compounds of formulas 1 to 6, and is not limited to aspecific one in so far as it has an apoptosis-inducing activity, acarcinostatic activity, antioxidant activities such as an activity ofinhibiting active oxygen production, an activity of inhibiting NOproduction, etc., and/or an immunoregulatory activity. Examples thereofinclude decomposition products of the raw substances such as productsfrom agarose obtained by decomposition with acid or digestion withα-agarase such as agarobiose, agarotetraose, agarohexaose, agarooctaose,agarodecaose, β-D-galacto-pyranosyl-3,6-anhydro-2-O-methyl-L-galactoseand the like. Furthermore, products from carrageenan obtained bydecomposition with acid or digestion with carrageenase such as carabiosecan also be exemplified. Furthermore, the saccharides of the presentinvention which contain the compounds selected from the compounds offormulas 1 to 6 at their reducing ends include those in which one ormore sugars selected from hexoses such as glucose, mannose, galactose,etc., pentoses such as xylose, arabinose, ribose, etc., uronic acidssuch as glucuronic acid, galacturonic acid, mannuronic acid, gluronicacid, etc., amino sugars such as glucosamine, galactosamine, etc.,sialic acids such as N-acetylneuraminic acid, etc., deoxy sugars such asfucose, etc., as well as esters, amides and lactones thereof are boundto hydroxy groups other than that at 1-position of the compoundsselected from the compounds of formulas 1 to 6. Furthermore, thesaccharides of the present invention which contain the compoundsselected from the compounds of formulas 1 to 6 at their reducing endsinclude those in which pyruvate and/or sulfate groups are bound to thesaccharides containing the compounds selected from the compounds offormulas 1 to 6 at their reducing ends, for example, the saccharidessuch as agarobiose, agarotetraose, agarohexaose, agarooctaose,K-carabiose, S-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose andthe like as well as the saccharides whose hydroxy groups are methylated.As described above, preferably, the saccharides of the present inventionwhich contain the compounds selected from the compounds of formulas 1 to6 at their reducing ends are those whose non-reducing ends are sugarsother than L-galactose-6-sulfate.

Since an anomer carbon is present at 1-position of the compound at thereducing end of the saccharide containing 3,6-anhydrogalactopyranose orits 2-O-methylated derivative at its reducing end, an α-isomer and aβ-isomer exist for such a compound. Both can be used as the saccharidesof the present invention which contain 3,6-anhydrogalactopyranose or its2-O-methylated derivative at their reducing ends.

The molecular weight is not specifically limited in so far as thecompound has a physiological activity such as an apoptosis-inducingactivity, a carcinostatic activity, antioxidant activities such as anactivity of inhibiting active oxygen production, an activity ofinhibiting NO production and/or an immunoregulatory activity.

Of course, a mixture of an α-isomer, β-isomer, an aldehyde and ahydrated, and a mixture of a D-isomer and a L-isomer can be used in thepresent invention as the compound selected from the compounds offormulas 1 to 6 or the saccharide containing the compound at itsreducing end.

Thus, the compound selected from the compounds of formulas 1 to 6 or thesaccharide containing the compound at its reducing end used in thepresent invention has an apoptosis-inducing activity, a carcinostaticactivity, antioxidant activities such as an activity of inhibitingactive oxygen production, an activity of inhibiting lipid peroxideradical production, an activity of inhibiting NO production, animmunoregulatory activity and an anti-allergic activity. Then, accordingto the present invention, first, there is provided a pharmaceuticalcomposition comprising as an active ingredient at least one of thecompounds selected from the compounds of formulas 1 to 6 and the solublesaccharides containing these compounds at their reducing ends fortreating or preventing a disease sensitive to at least one of thesecompounds, for example, a therapeutic or prophylactic composition forthe disease.

Examples of diseases sensitive to these compounds include a diseasesthat requires induction of apoptosis for its treatment or prevention, acarcinomatous disease, a diseases that requires inhibition of activeoxygen production for its treatment or prevention, a disease thatrequires inhibition of lipid peroxide radical production for itstreatment or prevention, a disease that requires inhibition of NOproduction for its treatment or prevention or a disease that requiresimmunoregulation for its treatment or prevention such as an allergicdisease. The pharmaceutical composition for treating or preventing thesediseases of the present invention can be used as a composition forinducing apoptosis, a carcinostatic composition, antioxidants such as aninhibitor of active oxygen production, an inhibitor of lipid peroxideradical production, an inhibitor of nitrogen monoxide production, ananti-inflammatory composition, an immunoregulator, an anti-allergiccomposition and the like.

For example, the composition for inducing apoptosis of the presentinvention is useful for eliminating auto-reactive lymphocytes frompatients suffered from autoimmune diseases, tumor cells, cells infectedwith a virus and the like. It can be used to eliminate unnecessary orharmful cells from a living body in a natural manner by causingapoptosis in the desired tissues or cells. Examples of diseases forwhich the composition for inducing apoptosis of the present invention iseffective include autoimmune diseases such as systemic lupuserythematosus, immune mediated glomerulonephritis, multiple sclerosis,collagen disease, etc., rheumatism, and the like.

The composition for inducing apoptosis of the present invention can beused in a method for inducing apoptosis and the method is useful forelucidation of mechanism of induction of apoptosis, as well as screeningfor apoptosis-inducing compounds and inhibitors of apoptosis induction.

Since the activity of inducing apoptosis by the composition for inducingapoptosis of the present invention is inhibited by Caspase inhibitor,for example, IL-LS converting enzyme inhibitor V[Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone: manufactured by TakaraShuzo]. Thus, the apoptosis induced by the composition is considered tobe a cell death due to apoptosis depending on Caspase.

Caspase has been shown that it functions as an important mediator ofapoptosis because it increases prior to various cell death; itsoverexpression induces cell death; the apoptosis is inhibited by apeptide inhibitor or an inhibitory protein such as CrmA and p35; and, ina knockout mouse for Caspase-1 or Caspase-3, a part of apoptosisnormally observed is inhibited [Seikagaku (Biochemistry), vol. 70, p.14-21 (1998)]. That is, during apoptosis process, Caspase which is acysteine protease is activated to decompose nuclear or cytoplasmicproteins. Caspase is first synthesized as a precursor and then activatedby processing. Regulation of this Caspase activation decides the life ordeath of cells. The mammals have 10 or more types of Caspases. Anupstream Caspase processes a downstream Caspase to amplify the activityof decomposing intracellular proteins in a cascade mode [Saibo Kogaku(Cell Technology), Vol. 17, p. 875-880 (1998)]. On the contrary, theprocessing activity can be inhibited by inhibitor of the cysteineprotease, Caspase, to stop cell death by Caspase dependent apoptosis.

The compound used in the present invention is useful for inhibition ofproduction of oxidizing materials such as active oxygen. Then, anantioxidant such as an inhibitor of active oxygen production whichcomprises the compound as its active ingredient is useful for treatingor preventing diseases caused by production and/or excess of activeoxygen.

In general, active oxygen can be classified into radical active oxygenand non-radical active oxygen. The radical active oxygen includeshydroxy radical, hydroxyperoxy radical, peroxy radical, alkoxy radical,nitrogen dioxide (NO₂), NO, thylradical and superoxide. On the otherhand, the non-radical active oxygen includes singlet oxygen, hydrogenperoxide, lipid hydroperoxide, hypochlorous acid, ozone andperoxonitrite. All of them are related to various pathological statessuch as inflammatory diseases, diabetes, cancers, arteriosclerosis,neurosis, ischemic re-perfusion disorder and the like.

In a living body, active oxygen is always produced at a lowconcentration in some pathways. These are superoxide physiologicallyleaking out from an electron transport system such as mitochondria,hydrogen peroxide, hydroxy radical catalyzed with a transition metalsuch as copper and iron, hypochlorous acid formed by neutrophils ormonocytes for protecting against infections, NO produced bydecomposition of arginine and the like, and they are inevitable. Aliving body has a system eliminating active oxygen including enzymes andlow molecular weight compounds against the production of active oxygento maintain the balance between the production and the elimination.However, a living body is damaged oxidatively when the system forproducing active oxygen becomes predominant over the eliminating systemdue to the activation of the above-mentioned pathways for some reasonsor, to the contrast, due to the inactivation of the eliminating system.Such conditions are called as oxidative stress. Furthermore, in additionto the imbalance inside the body, the living body is always exposed tooxidative stress by materials outside the body such as the atmosphere,foods and the like. Therefore, oxidative stress is inevitable ineveryone's daily life.

That is, as described above, the oxidative stress is related to variousdiseases and a living body is always exposed to circumstances in whichdiseases are caused by or disease conditions become more serious due tooxidative stress. Therefore, the antioxidant such as the inhibitor ofactive oxygen production of the present invention is useful forpreventing and treating the diseases caused by oxidative stress orpreventing the worsening of the disease conditions due to such oxidativestress.

Furthermore, a lipid peroxidation reaction is always associated withoxidative stress and proceeds at once upon production of a lipidperoxide radical. 4-Hydroxy-2-nonenal (HNE) produced therein is a toxicaldehyde specifically targeting glutathione or a protein. Reactionproducts of HNE and protein are detected in various disease tissues andconsidered to be inducing factors of disease conditions associated withoxidative stress. Then, the antioxidant which comprises the antioxidantsubstance used in the present invention which can inhibit production oflipid peroxide radicals is useful for preventing and treatingage-related diseases caused by oxidative stress.

NO is the main component of an endothelium-derived relaxing factor(EDRF) [Nature, Vol. 327, p. 524-526 (1987)]. According to the presentinvention, there is provided a pharmaceutical composition for treatingor preventing a diseases that requires inhibition of NO production forits treatment or prevention.

In the present invention, diseases that require inhibition of NOproduction are not limited to specific one and include for example,systematic hypotension caused by toxic shock, treatment with somecytokines and the like, blood pressure response reduction, autoimmunediseases, inflammation, arthritis, rheumatoid arthritis, diabetes,inflammatory bowel diseases, vascular function failure, pathogenicangiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, diseases associated with vascularization, cancers andthe like, inclusive the diseases described in JP-A 9-504524, JP-A9-505288, JP-A 8-501069, JP-A 8-512318 and JP-A 6-508849.

For NO synthases (NOS) which produces NO and L-citrulline from L-argnineand oxygen, a cNOS type which are constitutively expressed, and an iNOSwhich is a inducible type are known. In macrophages and the like, iNOSis induced by stimulation of cytotoxin or cytokines (for example, LPS,INF-γ) to produce NO. iNOS itself is essential to maintain a living bodysystem. However, on the other hand, it has been shown that iNOS causesvarious diseases when it is expressed excessively by various factors toproduce excess NO.

The present inventors have confirmed that the compounds selected fromthe compounds of formulas 1 to 6 and the soluble saccharides containingthese compounds at their reducing ends such as agarobiose, agarotetraoseand agarohexaose inhibit this iNOS expression. The confirmation wascarried out at protein level by western blotting and at messenger RNAlevel by RT-PCR. That is, the compounds used in the present inventionare useful for treating and preventing diseases that require inhibitionof NO production by inhibiting expression of iNOS which is overexpressedby various factors to produce excess NO.

The compounds of the present invention inhibit NO production inmacrophages and are useful for treating and preventing diseases causedby NO production in macrophages, inflammation, cancers and the like. Inaddition, inhibition of NO production by the saccharides used in thepresent invention is not antagonistic inhibition of NO productioninducing substances such as LPS or INF-γ. Increase in inhibitory effecton NO production is observed by addition of the saccharides used in thepresent invention in advance. Therefore, the compounds of the presentinvention are very useful as those for preventing antioxidantproduction.

The inhibitor of NO production of the present invention is useful forstudying the mechanism of NO production, and the mode of action of NOand can be used for screening of materials involved in the mechanism ofNO production.

Vascularization is necessary for growth of a solid cancer, and vascularendothelial growth factor/vascular permeability factor (VEGF) playimportant roles in this process. In various tumor cells, VEGF is inducedby NO. The inhibitor of NO production of the present invention alsoinhibits VEGF production of tumor cells by inhibiting NO production,thereby inhibiting vascularization around cancer tissues. When theinhibitor of NO production of the present invention is administered to amouse in which tumor cells have been transplanted subcutaneously to forma solid cancer, vascularization around the cancer tissue becomesinsufficient and the cancer falls out.

Nitrosoamines are a series of compounds in which nitro group is attachedto a secondary amine and several hundred types are known. Many of themshow carcinogenic activity to animals by damaging DNA. Nitrosoamines areconsidered to have a high relation to carcinogenesis of a human beingand usually produced by a reaction of a nitrite and an amine in astomach. NO also produces a nitrosoamine by reaction with an amine underphysiological conditions at a neutral pH range. NO production isaccelerated in a patient suffered from clonorchiasis or cirrhosis thathave a high relation to a cancer epidemiologically. Therefore, inparticular, carcinogenesis of a high-risk group can be prevented byadministration of the inhibitor of NO production of the presentinvention to prevent acceleration of NO production. As describedhereinabove, the inhibitor of NO production of the present inventionshows its carcinostatic activity in two step, that is, suppression ofcarcinogenesis and inhibition of vascularization in cancer tissues.

NO also induces edema which is specifically recognized in inflammatorylesions, i.e., vascular permeability accelerating activity [Maeda etal., Japanese Journal of Cancer Research, Vol. 85, p. 331-334 (1994)]and accelerates biosynthesis of prostaglandins which are inflammatorymediators [Salvemini et al., Proceedings of National Academy ofSciences, USA, Vol. 90, p. 7240-7244 (1993)]. On the other hand, NOreacts with a superoxide radical quickly to produce peroxonitrite ionand this peroxonitrite ion also considered to cause inflammatory damagesof cells and tissues.

NO production is induced when activated immune cells enter in an organand release cytokines. Insulin-dependent diabetes is induced by specificdestruction of islet β cells and this destruction is considered to becaused by NO. Synovial fluid in the lesion of a patient suffered fromrheumatoid arthritis, osteoarthrosis, gouty arthritis and arthritisassociated with Behçet disease contains NO at a concentration higherthan that in the normal joint of the same patient or joints of healthypeople. When the inhibitor of NO production of the present invention isadministered to such patients, NO production in the lesion is inhibitedto improve disease conditions.

NO production is increased during cerebral ischemia and afterre-perfusion, which causes damages in cerebral tissues. Administrationof the inhibitor of NO production of the present invention to a patientduring cerebral ischemia relieves the damage of cerebral tissue andimproves the prognosis.

The immunoregulator of the present invention has immunoregulatoryactivities such as an activity of suppressing lymphocyte blastogenesisand an activity of suppressing mixed lymphocyte reaction. Thus, theimmunoregulator of the present invention is useful as a pharmaceuticalcomposition for treating or preventing diseases caused by abnormality ofthese immune systems or immune factors.

Lymphocyte blastogenesis is a reaction in which mitogen binds to areceptor on the surface of a lymphocyte to activate the lymphocyte andpromotes its division and proliferation. Mixed lymphocyte reaction is areaction in which lymphocytes obtained from allogeneic animals are mixedand cultured, thereby inducing activation of lymphocytes due toincompatibility of major histocompatibility antigens to promote thedivision and proliferation of lymphocytes. The immunoregulator of thepresent invention suppress these reactions and is useful as apharmaceutical composition for treating and preventing chronic diseasescaused by abnormal acceleration of lymphocytes, for example, autoimmunediseases such as chronic nephritis, ulcerative colitis, type I diabetesand rheumatoid arthritis and is also useful for suppression of graftrejection.

In mast cells sensitized with IgE antibody, degranulation is induced bybinding of an antigen and a chemical mediator is released. This type I,i.e. the immediate-type allergic reaction plays an important role inallergy diseases whose representative examples are asthma and atopicdermatitis, and substances which suppress release of chemical mediatorsfrom mast cells are considered to be very effective for treating andpreventing these allergic diseases.

Passive cutaneous anaphylaxis (PCA) of a rat which is a model of thetype I allergic reaction is initiated with degranulation of mast cells,followed by release of chemical mediators contained in granules such ashistamine and serotonin to cause increase in vascular permeability andfinally to cause pigment leakage in a local skin. This model is used asa model for estimating anti-allergic compounds in vivo most frequently.

The compounds selected from the compounds of formulas 1 to 6 and thesoluble saccharides containing these compounds at their reducing endshas an activity of inhibiting PCA and the present invention alsoprovides an anti-allergic composition comprising at least one memberselected from the group consisting of the compounds selected from thecompounds of formulas 1 to 6 and the soluble saccharides containingthese compounds at their reducing ends as its active ingredient.

The anti-allergic composition is very useful for treating and preventingdiseases which can be treated by inhibition of the type I allergicreaction, such as bronchial asthma, atopic dermatitis, allergicrhinitis, pollinosis, hives, contact dermatitis, allergic conjunctivitisand the like.

The above-mentioned pharmaceutical composition for treating orpreventing diseases of the present invention, for example, thecomposition for inducing apoptosis, can be prepared by using at leastone member selected from the group consisting of the compounds selectedfrom the compounds of formula 1 to 6 and the soluble saccharidescontaining these compounds at their reducing ends as its activeingredient, and formulate it with a known pharmaceutically acceptablecarrier.

In general, the compound is combined with a pharmaceutically acceptableliquid or solid carrier and, if necessary, to this is added solvent,dispersing agent, emulsifier, buffering agent, stabilizer, excipient,binder, disintegrant, lubricant and the like to obtain a preparation inthe form of a solid preparation such as tablet, granule, powder,epipastic, capsule and the like, and a liquid preparation such as normalsolution, suspension, emulsion and the like. In addition, a driedpreparation which can be reconstituted as a liquid preparation byaddition of a suitable carrier before use can be obtained.

The composition for inducing apoptosis of the present invention can beadministrated as either an oral preparation or a parenteral preparationsuch as injectable preparation, drips or the like.

The pharmaceutical carrier can be selected according to theabove-mentioned particular administration route and dosage form. For anoral preparation, for example, starch, lactose, sucrose, mannit,carboxymethylcellulose, cornstarch, inorganic salts and the like areused. For preparing the oral preparation, binder, disintegrant,surfactant, lubricant, fluidity promoting agent, tasting agent, coloringagent, flavoring agent and the like can also be added.

A parenteral preparation can be prepared according to conventionalmethods by dissolving or suspending the active ingredient of the presentinvention, that is the saccharide having the activity of inducingapoptosis, in a diluent such as injectable distilled water,physiological saline, aqueous glucose solution, injectable vegetableoil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol,polyethylene glycol or the like, and, if necessary, adding sterilizer,stabilizer, osmotic regulator, smoothing agent and the like to theresultant solution or suspension.

The composition for inducing apoptosis of the present invention can beadministrated through a suitable route for the dosage form of thecomposition. The administration method is not limited and thecomposition can be used internally or externally (or topically) or byinjection and the like. The injectable preparation can be administratedintravenously, intramuscularly, subcutaneously, intradermally and thelike. External preparations include a suppository and the like.

A dosage of the composition for inducing apoptosis of the presentinvention can be appropriately determined and varies depending on theparticular dosage form, administration route and purpose as well as age,weight and conditions of a patient to be treated. In general, a dailydosage for an adult person is 10 μg to 200 mg/kg in terms of the amountof the active ingredient contained in the composition. As the dosage, ofcourse, can vary dependent on various factors, in some cases, a lessdosage than the above may be sufficient but, in other cases, a dosagemore than the above may be required. The pharmaceutical composition ofthe present invention can be administrated orally as it is, or it can beadministered daily by admixing with appropriate foods and drinks.

The carcinostatic composition of the present invention can be preparedby using at least one member selected from the group consisting of thecompounds selected from the compounds of formulas 1 to 6 and the solublesaccharides containing these compounds at their reducing ends as itsactive ingredient and formulating it with a known pharmaceuticalcarrier. The carcinostatic composition can prepared according to thesame manner as that described above with respect to the composition forinducing apoptosis.

The carcinostatic composition can be administrated through a suitableroute for the dosage form of the composition. A method foradministration is not limited and the composition can be administratedinternally or externally (or topically) or by injection and the like. Aninjectable preparation can be administrated, for example, intravenously,intramuscularly, subcutaneously, intradermally and the like. Externalpreparations include a suppository and the like.

A dosage of the carcinostatic composition of the present invention canbe determined and varies depending on the particular dosage form,administration route and purpose as well as age, weight and conditionsof a patient to be treated. In general, a daily dosage for an adultperson is 10 μg to 200 mg/kg in terms of the amount of the activeingredient contained in the composition. As the dosage, of course, canvary dependent on various factors, in some cases, a less dosage than theabove may be sufficient, but, in other cases, a dosage more than theabove may be required. The pharmaceutical composition of the presentinvention can be administrated orally as it is, or it can beadministrated daily by admixing with appropriate foods and drinks.

The antioxidant, the inhibitor of active oxygen production, theinhibitor of lipid peroxide radical production, the inhibitor of NOproduction, the immuno-regulator and the anti-allergic composition ofthe present invention can be prepared according to the same manner asthat described above with respect to the composition for inducingapoptosis. The same dosage and administration route as those describedabove with respect to the composition for inducing apoptosis can beused.

That is, the antioxidant, the inhibitor of active oxygen production, theinhibitor of lipid peroxide radical production, the inhibitor of NOproduction, the immuno-regulator and the anti-allergic composition ofthe present invention are administrated through a suitable route for theparticular dosage form of the composition. A method for administrationis not limited and the composition can be administrated internally orexternally, or by injection and the like. An injectable preparation canbe administrated intravenously, intramuscularly, subcutaneously,intradermally and the like. External preparations include a suppositoryand the like.

A dosage of the antioxidant, the inhibitor of active oxygen production,the inhibitor of lipid peroxide radical production, the inhibitor of NOproduction, the immuno-regulator and the anti-allergic composition ofthe present invention can be determined and varies depending on theparticular dosage form, administration route and purpose as well as age,weight and conditions of the patient to be treated. In general, a dailydosage for an adult person is 10 μg to 200 mg/kg in terms of the amountof the active ingredient contained in the composition. As the dosage, ofcourse, can vary dependent on various factors, in some cases, a lessdosage than the above may be sufficient, but, in other cases, a dosagemore than the above may be required. The pharmaceutical composition ofthe present invention can be administrated orally as it is, or it can beadministrated daily by admixing with appropriate foods and drinks.

The foods or drinks of the present invention are those comprising,produced by adding thereto and/or produced by diluting at least onemember selected from the group consisting of the compounds selected fromthe compounds of formulas 1 to 6 and the soluble saccharides containingthese compounds, for example, saccharides prepared by acid decompositionunder acidic conditions below pH 7 and/or enzymatic digestion of the rawsubstances, such as agarobiose, agarotetraose, agarohexaose,agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.Since the food or drink has an activity of inducing apoptosis, acarcinostatic activity, an antioxidant activity, an immunoregulatoryactivity and the like. Thus, it is very useful for ameliorating diseasestates of and preventing diseases sensitive to at least one memberselected from the group consisting of the compounds selected from thecompounds of formulas 1 to 6 and the soluble saccharides containingthese compounds at their reducing ends, such as a disease that requiresinduction of apoptosis for its treatment or prevention, a carcinomatousdisease, a disease that requires inhibition of active oxygen productionfor its treatment or prevention, a disease that requires inhibition ofNO production for its treatment or prevention or a disease that requiresimmunoregulation for its treatment or prevention, an allergic diseaseand the like.

A process for producing the foods or drinks of the present invention isnot limited to a specific one, and cooking, processing and othergenerally employed processes for producing foods and drinks can be usedin so far as the resultant foods or drinks contain as their activeingredients at least one member selected from the group consisting ofthe compounds selected from the compounds of formulas 1 to 6 and thesoluble saccharides containing those compounds at their reducing endsprepared, for example, by acid decomposition under acidic conditionsbelow pH 7 and/or enzymatic digestion of the raw substances, such asagarobiose, agarotetraose, agarohexaose, agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.

The foods or drinks of the present invention are not limited to aspecific one and examples thereof include cereal processed products(e.g., wheat flour products, starch processed products, premixedproducts, noodles, macaroni, breads, bean jams, buckwheat noodles, fu(wheat gluten bread), rice noodle, gelatin noodles, and packed ricecake, etc.), fat and oil processed products (e.g., plastic fat and oil,tempura oil, salad oil, mayonnaise, dressings, etc.), soybean processedproducts (e.g., tofu, miso, fermented soybeans, etc.), meet processedproducts (e.g., hams, bacon, pressed ham, sausage, etc.), processedmarine products (e.g., frozen ground fish meat, boiled fish paste,tubular roll of boiled fish paste, cake of ground fish, deep-fried pattyof fish paste, fish ball, sinew, fish meat ham, sausage, dried bonito,processed fish egg products, canned marine food, fish boiled insweetened soy sauce, etc.), dairy products (e.g., raw milk, cream,yoghurt, butter, cheese, condensed milk, powdered milk, ice cream,etc.), processed vegetables and fruit products (e.g., pastes, jams,pickles, fruit juices, vegetable drinks, mixed drinks, etc.),confectioneries (e.g., chocolates, biscuits, sweet buns, cakes,rice-cake sweets, rice sweets, etc.), alcohol drinks (e.g., sake,Chinese liquors, wines, whiskies, shochu, vodkas, brandies, gins, rums,beer, soft alcohol drinks, fruit liquors, liqueurs, etc.), luxury drinks(e.g., green tea, tea, oolong tea, coffee, soft drinks, lactic aciddrinks, etc.), seasonings (e.g., soy sauce, sauce, vinegar, sweet sake,etc.), canned food, bottled food and bagged food (e.g., various cookedfood such as rice topped with cooked beef and vegetables, rice boiledtogether with meat and vegetables in a small pot, steamed rice with redbeans, curry, etc.), semi-dried or condensed food (e.g., liver paste,the other spread, soup of buckwheat noodles or “udon”, condensed soups,etc.), dried food (e.g., instant noodles, instant curry, instant coffee,powdered juice, powdered soup, instant miso soup, cooked food, cookeddrinks, cooked soup, etc.), frozen food (e.g., sukiyaki, chawan-mushi,grilled eel, hamburger steak, shao-mai, Chinese meat dumpling, variousstick, fruit cocktail, etc.), solid food, liquid food (e.g., soup,etc.), processed agricultural products and forest products such asspices, processed livestock products, processed marine products and thelike.

In so far as the food or drink of the present invention comprises, isproduced by adding thereto and/or produced by diluting at least onemember selected from the group consisting of the compounds selected fromthe compounds of formulas 1 to 6 and the soluble saccharides containingthese compounds at their reducing ends, for example, saccharidesprepared by acid decomposition under acidic conditions below pH 7 and/orenzymatic digestion of the raw substances, such as agarobiose,agarotetraose, agarohexaose, agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like, inan amount necessary for exhibiting the physiological functions, theirforms are not limited to a specific one and may be any edible formsincluding tablets, granule, capsule and the like.

3,6-Anhydrogalactopyranose, a 2-O-methylated derivative thereof and thesaccharides containing these compounds at their reducing ends tend toopen at their hemi-acetal rings to form aldehyde groups at the ends.These aldehyde groups as well as the aldehyde group of the aldehyde ofthe 3,6-anhydrogalactopyranose tend to react with compounds which arereactive with aldehyde group, for example, nucleophiles such as aminoacids. The compounds of formulas 1 to 6 or the saccharides, for example,the oligosaccharides thus reacted are in such a state that they lose thecompounds selected from the compounds of formulas 1 to 6 at theirreducing ends. Therefore, they lose various physiological activities ofthe member selected from the compounds selected from the compounds offormulas 1 to 6 and the oligosaccharides containing these compounds attheir reducing ends. That is, in order to maintain the member selectedfrom the compounds selected from the compounds of formulas 1 to 6 andthe saccharides containing these compounds at their reducing ends in thefoods or drinks stably, a molar concentration of a compound reactivewith the aldehyde should be kept lower than that of the aldehyde.

In the production of the food or drink of the present invention, it ispossible to provide the food or drink that contains the member selectedfrom the group consisting of the compounds selected from the compoundsof formulas 1 to 6 and the oligosaccharides containing these compoundsat their reducing ends in a high content without substantial reductionof the amount thereof by controlling the amount of a compound that isreactive with the aldehyde. Such control has not been consideredheretofore in the prior art.

It is also found that the member selected from the group consisting ofthe compounds selected from the compounds of formula 1 to 6 and thesaccharides containing these compounds at their reducing ends is stableunder acidic conditions. Then, an acidic food or acidic drink whichcontains at least one member selected from the group consisting of thecompounds selected from the compounds of formulas 1 to 6 and solublesaccharide containing these compounds at their reducing ends in a highcontent can be provided by carrying out all of the steps of producingthe food or drink of the present invention under acidic conditions toprepare the acidic food or acidic drink.

In the production of the acidic food or drink of the present invention,the kind of the acid to be used for acid decomposition of the rawsubstances is not limited to a specific one, and both organic andinorganic acids can be used. However, a better taste of the resultantacid decomposition product of agar is obtained when an organic acid areused. Then, organic acids are preferably used to obtain an aciddecomposition product having a novel flavor. The organic acid can beselected depending on the particular purpose. It can be used alone, ortwo or more of them can be used in combination. Preferred examples ofthe organic acids include acetic acid, citric acid, malic acid, lacticacid, tartaric acid, succinic acid, fumaric acid and the like.Decomposition conditions are not specifically limited. For example, whencitric acid is used as the organic acid, acid decomposition of agar as araw substance is carried out at 60 to 130° C., preferably 90 to 105° C.,for 3 to 300 minutes, preferably 30 to 200 minutes, thereby modifying anacidic taste of the organic acid to obtain the composition having a goodbalanced taste, mild texture and a smooth acidic taste. An acidulanthaving the desired acidic taste can be obtained by heat treatment of acomposition containing 0.05 to 30% by weight, preferably 0.2 to 10% byweight of an organic acid, and 1 to 20% by weight, preferably 5 to 15%by weight of at least one member selected from the group consisting ofthe compounds selected from the compounds of formulas 1 to 6 and thesoluble saccharides containing these compounds at their reducing ends.The acidulant thus obtained is very useful for the production of softdrinks, acidic seasonings, acidic foods and the like.

The acidic food or acidic drink of the present invention contains alarge amount of at least one member, which has a physiological activity,selected from the group consisting of the compounds selected from thecompounds of formulas 1 to 6 and the soluble saccharides containingthese compounds, for example, saccharides prepared by acid decompositionunder acidic conditions below pH 7 and/or enzymatic digestion of the rawsubstances, such as agarobiose, agarotetraose, agarohexaose,agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.The physiological functions of the compounds such as an activity ofinducing apoptosis, a carcinostatic activity and the like provide aneffect of preventing carcinogenesis, an effect of suppressing cancer orthe like upon eating the food or drink. That is, the acidic food oracidic drink of the present invention is a healthy food or drink whichhas effects of ameliorating the disease states of or preventing thediseases sensitive to at least one member selected from the groupconsisting of the compounds selected from the compounds of formulas 1 to6 and the soluble saccharides containing these compounds, and isparticularly useful for keeping gastrointestinal health.

The compounds selected from the compounds of formulas 1 to 6 and thesoluble saccharides containing said compounds of the present invention,for example, saccharides prepared by acid decomposition under acidicconditions below pH 7 and/or enzymatic digestion of the raw substances,such as agarobiose, agarotetraose, agarohexaose, agarooctaose,κ-carabiose, β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose andthe like have antioxidant activities such as an activity of inhibitingactive oxygen production, an activity of inhibiting lipid peroxideradical production and the like, and can be used in the productionantioxidant foods or antioxidant drinks as an antioxidant such as aninhibitor of active oxygen production, an inhibitor of lipid peroxideradical production, an inhibitor of NO production and the like.

That is, according to the present invention, there is provided anantioxidant, in particular, an antioxidant for foods and drinks, whichcomprises at least one member selected from the group consisting of thecompounds selected from the compounds of formulas 1 to 6 and the solublesaccharides containing these compounds as its active ingredient.

The form of the antioxidant of the present invention is not limited to aspecific one, and can be suitably selected according to the foods anddrinks to be applied, for example, powder, paste, emulsion and the like.The antioxidant food or drink which comprises the member selected fromthe compounds and the saccharides used in the present invention as itsactive ingredient can be readily and simply produced by using theantioxidant of the present invention.

According to the present invention, there is also provided a saccharidefor an antioxidant selected from the group consisting of the compoundsselected from the compounds of formulas 1 to 6 and the solublesaccharide containing these compounds. For example, the saccharide foran antioxidant can be obtained as a product produced by acid decomposedunder acidic conditions below pH 7 and/or enzymatic digestion of the rawsubstance. In addition, its purified or partial purified product canalso be used. Examples of the raw substances include those derived fromred algae such as agar, agarose, carrageenan and the like. They can beused alone or two or more of them can be used in combination. Theexamples of representative saccharides for an antioxidant are, notlimited specifically, soluble polysaccharides containing the compoundsselected from the compound of formulas 1 to 6, for example, agarobiose,agarotetraose, agarohexaose, agarooctaose, K-carabiose andβ-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.

The saccharide for an antioxidant of the present invention is useful foreliminating or suppressing the production of oxidants in a living body,such as active oxygen. Then, the saccharide for an antioxidant is usefulfor ameliorating disease states of or preventing diseases caused byproduction or excess of active oxygen.

As described above, oxidative stress, which is generated from oxidativedamage of a living body in case where the system for producing activeoxygen is predominant over an elimination system, is involved in variousdiseases. Thus, a living body is always exposed to circumstances whichlead to diseases caused by oxidative stress or worsening of the diseasesconditions. Therefore, it is desirable to take a suitable amount of anantioxidant everyday for preventing, treating or preventing worsening ofdiseases caused by oxidative stress. For daily intake of suitable amountof an antioxidant, it is desirable to take it from foods and drinks. Thefoods and drinks of the present invention which comprise, produced byadding thereto, and/or produced by adding the saccharide for anantioxidant are very useful for antioxidant foods or drinks oranti-oxidative stress foods or drinks.

The member selected from the compounds and the saccharides used in thepresent invention also have ability of retaining water and at least oneof them can be used as an active ingredient for the production of ananti-constipation composition, an anti-constipation food and ananti-constipation drink.

Furthermore, according to the present invention, there is provided acosmetic composition which comprises as its active ingredient thesoluble saccharide containing the compound selected from the compoundsof formulas 1 to 6 in its reducing end, for example, an oligosaccharidesuch as agarobiose, agarotetraose, agarohexaose, agarooctaose,κ-carabiose, β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose orthe like. The saccharide can be obtained as a product produced by aciddecomposition under acidic conditions below pH 7 and/or enzymaticdigestion of the raw substance. The purified or partially purifieddecomposition product can also be used. As the raw substance, thatderived from red algae, for example, agar, agarose, carrageenan or thelike can be used alone or two or more of them can be used incombination.

The above-mentioned compound can be used as an active ingredient for theproduction of cosmetic compositions including fundamental cosmeticcompositions such as cream, milky lotion, lotion, facial cleansing andpuck, makeup cosmetics such as lipstick and foundation, body soap, soapand the like. The compound is also effective to the hair and thecosmetic composition of the present invention can be produced in theform of hair care products, for example, hair products such as hairtonic, hair liquid, hair set lotion, hair blow agent, hair cream, haircoat, and the like and hair toiletry products such as shampoo, hairrinse, hair treatment, and the like. The amount of the compound mixed inthe cosmetic composition can be determined appropriately according toits skin beautifying/whitening activity, humectant or moisturizingactivity, antioxidant activity and the like. As other cosmeticcomponents, those mixed in conventional cosmetic compositions can beused. Skin beautifying/whitening activity and humectant or moisturizingactivity can be measured by conventional methods, for example the methoddescribed in JP-A 8-310937.

The cosmetic composition of the present invention has excellentproperties based on a skin beautifying/whitening activity, a humectantor moisturizing activity, an antioxidant activity, an activity ofinhibiting active oxygen production and an anti-oxidative stressactivity to the skin; a humectant or moisturizing activity, anantioxidant activity, an activity of inhibiting active oxygen productionand an anti-oxidative stress activity to the hair; and the like.

The present invention also provides a preservative composition forkeeping freshness of foods and drinks which comprises as its activeingredient at least one member selected from the group consisting of thecompounds selected from the compounds of formulas 1 to 6 and the solublesaccharides containing these compounds at their reducing ends, forexample, oligosaccharides such as agarobiose, agarotetraose,agarohexaose, agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like.The saccharide can be obtained as a product produced by aciddecomposition under acidic conditions below pH 7 and/or enzymaticdigestion of the substance. The purified or partially purifieddecomposition product also can be used. As the raw substance, thatderived from red algae which comprises the compound selected from thecompounds of formulas 1 to 6, for example, agar, agarose, carrageenanand the like can be used alone or two or more can be used incombination.

At least one member selected from the group consisting of the compoundsselected from the compounds of formulas 1 to 6 and the solublesaccharides containing the compounds at their reducing ends, forexample, a saccharide such as agarobiose, agarotetraose, agarohexaose,agarooctaose, κ-carabiose,β-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and the like hasan antioxidant activity, a freshness keeping activity and a tyrosinaseinhibitory activity. The preservative composition for keeping freshnessof foods and drinks of the present invention which prevents effectivelycolor change, decay, oxidation and the like of foods can be produced byusing the compound as its active ingredient according to a knownformulation process. The preservative composition of the presentinvention is very useful for keeping a flavor and freshness of variousfoods, perishable foods, and processed foods.

No acute toxicity is observed when administering either of the memberselected from the group consisting of the compounds selected from thecompounds of formula 1 to 6 and the soluble saccharides containing thesecompounds used in the present invention to a mouse at a dosage of 1 g/kgorally or intraperitoneally.

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

Example 1

(1) A suspension of 400 mg of commercially available agar powder(manufactured by Wako Pure Chemical Industries, Ltd.) in 20 ml of 1 NHCl was heated with a microwave oven to obtain a solution. The resultingsolution was cooled and was adjusted to pH 4 with sodium hydroxide. Tothe solution was added 384 mg of citric acid and the solution wasadjusted to pH 3 with sodium hydroxide. Water was added thereto to makethe total volume up to 40 ml and the solution was heated at 120° C. for4 hours. The resulting acid decomposition solution was adjusted to pH6.5 with sodium hydroxide and filtrated with a 0.2 μm filter(manufactured by Corning).

Human promyelocytic leukemia cell HL-60 (ATCC CCL-240) was incubated at37° C. in RPMI 1640 medium (manufactured by Gibco) supplemented with 10%of fetal bovine serum (JRH Bioscience) which had been treated at 56° C.for 30 minutes, and suspended in the same medium at a concentration of500 cells/90 μl. Each 90 μl portion of the suspension was distributedinto each well of a 96 well plate (manufactured by Falcon). To thesuspension in each well was added 10 μl of the above-mentioned aciddecomposition solution, a 10-fold dilution of the solution or water, andincubated with 5% CO₂ at 37° C. After 24 hour and 48 hour from theinitiation of the incubation, the cell morphology was observed under anoptical microscope. Then, according to the MTT method described in“Apoptosis Jikken Protocol” (Syuzyun-sha, Tanuma, Seiichi ed., pp. 156(1994)), 5 mg/ml of3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide(manufactured by Sigma) and 10 μl of phosphate buffered saline solutionwere added to the culture and the incubation was continued foradditional 4 hours. Then, 100 μl of 2-propanol containing 0.04 Nhydrochloric acid was added to the culture, and the mixture wasthoroughly stirred. An absorbance at 590 nm was measured and the numberof viable cells was calculated from the absorbance measured for each ofthe wells, which was compared each other.

Example 2

(1) A suspension of 1 g of commercially available agar (Agar Noble,manufactured by Difco) in 100 ml of 0.1 N hydrochloric acid was heatedwith a microwave oven until boiling to prepare a solution. After coolingto room temperature and adjusting to pH 6, the solution was filteredthrough Cosmonice filter (manufactured by Nacalai Tesque) and 2 ml ofthe filtrate was separated with reverse phase HPLC under the followingconditions.

-   -   Column: TSK-gel ODS 80Ts (20 mm×250 mm, manufactured by Toso)    -   TSK guard column ODS-80Ts □20 mm×50 mm, manufactured by Toso)    -   Mobile phase: aqueous 0.1% trifluoroacetic acid (TFA) solution    -   Flow rate: 9 ml/min    -   Detection: absorbance at 215 nm

Each elution peak was fractionated, collected, evaporated to drynessunder reduced pressure and then dissolved in 300 μl of water. Eachfraction was sterilized by filtration and its 10 μl portion was placedin a well of a 96 well microtiter plate. Then, 90 μl of RPMI 1640 medium(manufactured by Nissui) containing 10% fetal bovine serum (manufacturedby Gibco) and 5,000 HL-60 cells (ATCC CCL-240) was added thereto,followed by incubation at 37° C. for 48 hours with 5% CO₂. The cellmorphology was observed under an optical microscope. Then, 5 mg/ml3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and 10 μlof phosphate buffered saline solution were added thereto and theincubation was continued for additional 4 hours. To the culture wasadded 100 μl of 2-propanol containing 0.04 N hydrochloric acid and theresultant mixture was thoroughly stirred. The absorbance at 590 nm wasmeasured to determine a cell proliferation rate.

As a result, apoptosis corpuscles were observed in the group to whichthe fraction from the peak at 8.26 min. was added. And, as compared withthe control group to which water was added, the absorbance at 590 nm waslower and the cell proliferation was inhibited.

(2) A 100 μl of the fraction from the peak at 8.26 min. described inExample 2-(1) was separated with size exclusion HPLC chromatography asfollows.

-   -   Column: TSK-gel α-2500 (7.8 mm×300 mm, manufactured by Toso)    -   TSK guard column a (6 mm×40 mm, manufactured by Toso)    -   Mobile phase: aqueous 0.01% TFA solution    -   Flow rate: 0.8 ml/min    -   Detection: differential refractometer

The separation pattern of the size exclusion HPLC chromatography isillustrated in FIG. 3. That is, FIG. 3 illustrates the size exclusionHPLC chromatogram of the acid decomposition product of agar. Thehorizontal axis represents the elution time (min.) and the vertical axisrepresents the output from the differential refractometer.

Each separated peak was fractionated, collected and evaporated todryness under reduced pressure. Each fraction was dissolved in water ata concentration of 10 mg/ml, sterilized by filtration and then,according to the same manner as that described above in Example 2-(1),an apoptosis-inducing activity and an antiproliferation activity againsttumor cell were measured. As a result, the peaks at the elution time8.87 min. and 9.40 min. had both activities.

The substances at the elution time 8.87 min. and 9.40 min. wereseparately dissolved in a phosphate buffered saline solution, andallowed to stand at 37° C. for 1 hour. Then, according to the samemanner as that described above, they were analyzed by the size exclusionHPLC chromatography. As a result, the peaks at 8.87 min. and 9.40 min.were observed in both samples, and the ratio of the peak area was almostidentical among the samples. This revealed that the substances at theelution time 8.87 min. and 9.40 min. were in an equilibrium state whenthey were dissolved in the aqueous phosphate buffer.

The fractions from the peaks at the elution time 8.87 min. and 9.40 min.were combined and evaporated to dryness under reduced pressure to obtainan apoptosis-inducing and carcinostatic substance.

(3) The apoptosis-inducing and carcinostatic substance described inExample 2-(2) was subjected to mass spectrometry with DX302 massspectrometer (manufactured by Nippon Denshi). The measurement wascarried out by using glycerol as a matrix with negative ion mode.

FAB-MS

m/z 323 [M-H]⁻

415 [M+glycerol-H]⁻

507 [M+2glycerol-H]⁻

The results are shown in FIG. 4. That is, FIG. 4 illustrates the massspectrum of the apoptosis-inducing and carcinostatic substance. Thehorizontal axis represents m/z value and the vertical axis representsthe relative intensity (%).

Nuclear magnetic resonance spectrum of the apoptosis-inducing andcarcinostatic substance described in Example 2-(2) was measured withJNM-A500 nuclear magnetic resonance apparatus (manufactured by NipponDenshi).

¹H-NMR: δ 3.36 (1H, dd, J=8.0, 10.0 Hz), 3.51 (1H, dd, J=3.0, 10.0 Hz),3.56 (1H, m), 3.58 (1H, m), 3.63 (1H, m), 3.67 (1H, m), 3.70 (1H, dd,J=3.0, 10.0 Hz), 3.77 (1H, d, J=3.0 Hz□, 3.83 (1H, dd, J=4.5, 10.0 Hz),3.93 (1H, dd, J=5.0, 3.5 Hz), 4.23 (1H, m), 4.25 (1H, m), 4.41 (1H, d,J=8.0 Hz), 4.85 (1H, d, J=6.0 Hz)

The sample was dissolved in heavy water and the chemical shift value ofHOD was shown as 4.65 ppm.

¹³C-NMR: δ 61.9, 69.4, 71.5, 73.37, 73.42, 73.8, 76.0, 76.1, 83.7, 86.5,90.7, 103.3

The sample was dissolved in heavy water and the chemical shift value ofdioxane was shown as 67.4 ppm.

¹H-NMR spectrum of the apoptosis-inducing and carcinostatic substance isshown in FIG. 5. In FIG. 5, the horizontal axis represents the chemicalshift value (ppm) and the vertical axis represents the signal intensity.

The sample was also dissolved in heavy dimethyl sulfoxide and ¹H-NMRspectrum was measured.

¹H-NMR: δ 9.60 (1H, H of aldehyde)

¹H-NMR spectrum of the apoptosis-inducing and carcinostatic substance inheavy dimethyl sulfoxide solvent is shown in FIG. 6. In FIG. 6, thehorizontal axis represents the chemical shift value (ppm) and thevertical axis represents the signal intensity.

The apoptosis-inducing and carcinostatic substance described in Example2-(2) was identified as agarobiose on the basis of the analyticalresults of mass spectrometry, ¹H-NMR and ¹³C-NMR. And, the ¹H-NMR inheavy dimethyl sulfoxide solvent demonstrated that 3,6-anhydrogactose atthe reducing end of agarobiose was mainly present as an aldehyde whosering was opened in a non-aqueous solvent. In addition, ¹H-NMR in heavywater solvent demonstrated that it was present as a hydrated of thealdehyde in an aqueous solution.

The results as described above revealed that the apoptosis-inducing andcarcinostatic substance obtained in Example 2-(2) was agarobiose.

(4) The apoptosis-inducing and carcinostatic substance obtained inExample 2-(2), i.e., agarobiose, was dissolved in water at aconcentration of 0.78 mg/ml, and its 10 μl portion was placed in thewell of a 96 well microtiter plate to measure an apoptosis-inducingactivity and an antiproliferation activity against cell according to thesame manner as described in Example 2-(1). As a result, apoptosiscorpuscles were observed under an optical microscope and, as comparedwith the control group to which water as added, cell proliferation wassuppressed by about 86% in the group to which agarobiose was added.Namely, agarobiose at a concentration of 78 μg/ml induced apoptosis inHL-60 cells and inhibited cell proliferation.

Example 3

(1) A suspension of 2.5 g of commercially available agar (Agar Noble) in50 ml of 0.1 N HCl was heated at 100° C. for 13 minutes to prepare asolution. After cooling to room temperature and neutralizing to aboutneutral pH with NaOH, the solution was filtered through Cosmonice filterand separated with normal phase HPLC as follows.

Column: TSk-gel Amide-80 (21.5 mm×300 mm, manufactured by Toso)

Solvent A: aqueous 90% acetonitrile solution

Solvent B: aqueous 50% acetonitrile solution

Flow rate: 5 ml/min

Elution: linear gradient from solvent A to solvent B (80 min.)>Solvent B(20 min.)

Detection: absorbance at 195 nm

Amount of sample applied: 2 ml

The peaks at the retention time 66.7 min., 78.5 min. and 85.5 min. werefractionated and collected and they were subjected to mass spectrometry.As a result, these substances were agarobiose, agarotetraose andagarohexaose, respectively. The separation with HPLC as described abovewas repeated 8 times and the fractions thus separated were evaporated todryness under reduced pressure to obtain 122 mg of agarobiose, 111 mg ofagarotetraose, and 55 mg of agarohexaose, respectively.

The results are shown in FIGS. 7 to 10. That is, FIG. 7 illustrates theelution pattern of agarobiose, agarotetraose and agarohexaose in thenormal phase HPLC. The horizontal axis represents the retention time(min.) and the vertical axis represents the absorbance at 195 nm. FIG. 8illustrates the mass spectrum of the peak at 66.7 min. The horizontalaxis represents the m/z value and the vertical axis represents therelative intensity (%). FIG. 9 illustrates the mass spectrum of the peakat 78.5 min. The horizontal axis represents the m/z value and thevertical axis represents the relative intensity (%). FIG. 10 illustratesthe mass spectrum of the peak at 88.5 min. The horizontal axisrepresents the m/z value and the vertical axis represents the relativeintensity (%).

(2) To 450 μl of 100 mM aqueous agarobiose solution obtained in Example3-(1) were added 50 μl of 10-fold concentrated phosphate buffered saline(T900, manufactured by Takara Shuzo) and 50 μl of 10 units/μl ofβ-galactosidase (G5635, manufactured by Sigma) in phosphate bufferedsaline. The resultant mixture was incubated at 37° C. for 1 hours.

To the reaction mixture was added 5 ml of a mixture of1-butanol:ethanol=1:1 and then insoluble materials were removed bycentrifugation. The resultant solution was applied on silica gelBW-300SP for column chromatography (3×50 cm, manufactured by FujiSilysia Chemical Ltd.) and separated using 1-butanol:ethanol:water=5:5:1as the eluent with pressurizing at 0.3 kg/cm² with a compressor.Fractionation was carried out to collect 7 ml fractions, and a portionof each fraction was taken up and analyzed with thin layerchromatography. As a result, Fraction Nos. 14 to 17 contained3,6-anhydro-L-galactose with high purity. These fractions were combinedand evaporated to dryness under reduced pressure to obtain 3.8 mg of3,6-anhydro-L-galactose. The structure of this substance was confirmedby mass spectrometry and nuclear magnetic resonance.

The results are shown in FIGS. 11 to 13. That is, FIG. 11 illustratesthe mass spectrum of 3,6-anhydro-L-galactose. The horizontal axisrepresents the m/z value and the vertical axis represents the relativeintensity (%). FIG. 12 illustrates the ¹H-NMR spectrum of3,6-anhydro-L-galactose in heavy water and FIG. 13 illustrates the¹H-NMR spectrum of 3,6-anhydro-L-galactose in heavy dimethyl sulfoxidesolvent. In the figures, the horizontal axes represent the chemicalshift value, and the vertical axes represent the signal intensity.

For 3,6-anhydro-L-galactose, ¹H-NMR spectrum in heavy dimethyl sulfoxidesolvent also showed the proton signal of aldehyde at 9.60 ppm. Thisdemonstrated that it was present as an aldehyde whose ring was opened ina non-aqueous solvent. Furthermore, from the ¹H-NMR spectrum in heavywater, it was present as a hydrate of the aldehyde in an aqueoussolution.

Example 4

(1) A 20 mM solution of 3,6-anhydro-L-galactose obtained in Example 3was diluted 2-, 4- and 8-folds with sterilized water and, according tothe same manner as that described in Example 2-(1), anapoptosis-inducing activity and an anti-proliferation activity againsttumor cells of respective dilutions were measured. As a result, in thegroup to which the 2-fold dilution of 3,6-anhydro-L-galactose was added(at the final concentration of 1 mM), apoptosis corpuscles were observedand the absorbance at 590 nm became less than one-half of that of thecontrol group to which water was added.

(2) A 50 mM solution of agarobiose, agarotetraose or agarohexaoseobtained in Example 3-(1) was sterilized by filtration and diluted 2-,4-, 8-, 16-, 32-, 64- and 128-folds with sterilized water. According tothe same manner as that described in Example 2-(1), ananti-proliferation activity against various cells of the resultantdilutions was measured. The cells and culture media used are shown inTables 1 and 2. TABLE 1 Cells Medium Human promyelocytic leukemia RPMI1640 medium (Nissui) HL-6O (ATCC CCL 240) supplemented with 10% fetalbovine serum (Gibco) Human peripheral lymphocyte the same as the aboveRPMI 1778 (ATCC CCL 156) Mouse monocyte DMEM medium supplemented RAW264.7 (ATCC TIB 71) with 10% fetal bovine serum (Nissui) Human gastriccancer cell RPMI 1640 medium MKN 45 (Riken gene bank, RCB supplementedwith 10% fetal 1001) bovine serum Human hepatoma cancer cell DMEM mediumsupplemented HepG2 (ATCC HB 8065) with 10% fetal bovine serum Humancolonic adenocarcinoma McCoy's medium supplemented HT-29 (ATCC HTB 38)with 10% fetal bovine serum (BioWhittaker) Human colonic adenocarcinomathe same as the above HCTll6 (ATCC CCL-247) Fibrosarcoma DMEM mediumsupplemented HP-1080 (ATCC CCL-121) with 10% fetal bovine serum(BioWhittaker)

TABLE 2 Cells Medium Glial blast cell DMEM medium supplemented A-172(ATCC CRL1620) with 10% fetal bovine serum Human breast cancer DMEMmedium supplemented MCF7 (ATCC HTB-22) with 10% fetal bovine serum Humanbreast cancer RPMI 1640 medium T-47D (ATCC HTB-133) supplemented with10% fetal bovine serum Human bladder carcinoma McCoy's mediumsupplemented T24 (ATCC HTB-4) with 10% fetal bovine serum Human cancerof the uterine DMEM medium supplemented cervix cell with 10% fetalbovine serum HeLa S3 (ATCC CCL-22) Human lung cancer the same as theabove A549 (ATCC CCL-185) Human colonic adenocarcinoma RPMI 1640 mediumWiDr (ATCC CCL-218) supplemented with 10% fetal bovine serum

As a result, agarobiose, agarotetraose and agarohexaose exhibited anantiproliferation activity against these cells. The results are shown inTable 3.

The number in Table 3 represents the dilution rate of the dilution addedto the group whose absorbance at 590 nm was less than one-half of thatof the control group to which water was added. The dilution rate 1corresponds to the concentration of 5 mM in the cell culture medium.Then, for agarobiose, agarotetraose and agarohexaose, the concentrationsrequired for 50% proliferation inhibitory rate (IC₅₀) are calculatedbased on the change in the absorbance at 590 nm, and are shown in Table4. TABLE 3 Cells Agarobiose Agarotetraose Agarohexaose HL-60 32 64 64RPMI1788 64 128 128 RAW264.7 16 32 32 MKN45 8 16 16 HepG2 8 8 16 HT-29 816 32 HCT116 16 32 32 HT-1080 8 16 16 A-172 16 16 16 MCF7 16 16 16 T-47D16 16 16 T24 8 8 8 HeLa S3 8 8 16 A549 8 8 16 WiDr 8 8 16

TABLE 4 IC₅₀ (μM) Cells Agarobiose Agarotetraose Agarohexaose HL-60 17097 78 RPMI1788 44 28 22 RAW264.7 179 133 109 MKN45 344 196 166 HepG2 652413 430 HT-29 622 208 144 HCT116 244 158 136 HT-1080 317 216 185 A-172289 185 151 MCF7 274 276 238 T-47D 210 183 158 T24 352 399 365 HeLa S3353 400 334 A549 570 494 279 WiDr 405 399 334

Example 5

A suspension of 5 g of commercially available agar in 50 ml of 0.1 N HClwas heated at 100° C. for 13 minutes. After cooling to room temperature,the solution was neutralized to about neutral pH with NaOH and 2 ml ofthe solution was applied to a column (10×255 mm) packed with activatedcarbon (60-150 mesh, 079-21, manufactured by Nacalai Tesque) washed withwater. The column was washed with 200 ml of water and then eluted witheach 200 ml of 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%,27.5%, 30%, 35%, 40%, 45% and 50% aqueous ethanol in this order.

Each eluted fraction was concentrated 10-folds under reduced pressureand spotted on a silica gel sheet 60F₂₅₄ (manufactured by Merck) anddeveloped with 1-butanol:ethanol:water=5:5:1. Orcinol reagent [preparedby dissolving 400 mg of orcinol monohydrate (manufactured by NacalaiTesque) in 22.8 ml of sulfuric acid and adding water thereto to make thefinal volume up to 200 ml] was sprayed to observe the resultant spots.

As a result, agarobiose with high purity was contained in the fractionseluted with 5% and 7.5% aqueous ethanol; agarotetraose with high puritywas contained in the fractions eluted with 15% and 17.5% aqueousethanol; agarohexaose with high purity was contained in the fractionseluted with 22.5% and 25% aqueous ethanol; and agarooctaose with highpurity was contained in the fractions eluted with 27.5% and 30% aqueousethanol.

Example 6

Agarobiose, agarotetraose, agarohexaose and agaro-octaose (hereinafter,sometimes, these oligosuccharaides are referred to asagarooligosaccharides) obtained in Example 5 were dissolved in water ata concentration of 2.5 mM or 1.25 mM separately, and were sterilized byfiltration. HL-60 cells were suspended in RPMI 1640 medium containing10% fetal bovine serum at a concentration of 2.5×10⁵ cells/4.5 ml and tothis suspension was added 0.5 ml of each of the oligosaccharidesolutions. The resultant mixture were incubated with 5% CO₂ at 37° C.for 24 hours or 48 hours. A part of the cell culture was taken up,followed by addition of Trypan Blue thereto and observing under amicroscope to count the number of viable cells. As a result, the numberof viable cells in each group was decreased as compared with the groupto which water was added (control) and apoptosis corpuscles wereobserved.

The results are shown in FIGS. 14 and 15. That is, FIG. 14 illustratesthe relation between the incubation time and the number of viable cellswhen HL-60 cells were cultured with addition of one of theoligosaccharides at the final concentration of 250 μM. FIG. 15illustrates the relation between the incubation time and the number ofviable cells when HL-60 cells were cultured with addition of one of theoligosaccharides at the final concentration of 125 μM. In FIGS. 14 and15, the horizontal axes represent the incubation time (hrs.) and thevertical axes represent the number of viable cells (×10⁵/5 ml). Theclosed circle (●) represents the addition of water (control), the opendiamond (⋄) represents the addition of agarobiose, the open circle (◯)represents the addition of agarotetraose, the open triangle (Δ)represents the addition of agarohexaose and the open square (□)represents the addition of agarooctaose.

Example 7

(1) A suspension of 0.2 g of K-carrageenan (manufactured by Sigma,C-1263) or λ-carrageenan (manufactured by Wako Pure Chemical Industries,Ltd., 038-14252) in 20 ml of 0.1 N HCl was heated at 95° C. for 10minutes. After neutralizing with 1N NaOH, the resultant mixture wasdiluted 1.5-, 2.25-, 3.38- and 5.06-folds with water and ananti-proliferation activity against HL-60 cells was measured accordingto the same manner as that described in Example 2-(1). As a result, inthe groups to which the 1.5-, 2.25- and 3.38-fold dilutions of heatedK-carrageenan and the 1.5- and 2.25-fold dilutions of heatedλ-carrageenan were added, the absorbance at 590 nm was less thanone-half of that of the control group to which water was added, andapoptosis corpuscles were observed.

(2) Commercially available agar (Agar Noble), agarose L03 (manufacturedby Takara Shuzo) and commercially available bar-shaped agar weresuspended in 1N HCl at a concentration of 1%, respectively, and heatedat 100° C. for 15 minutes. After cooling, the heated mixtures wereneutralized with 1N NaOH and diluted 2-, 4-, 8- and 16-folds with water,respectively. An anti-proliferation activity against HL-60 cells of theresultant dilutions were measured according to the same manner as thatdescribed in Example 2-(1). As a result, in the groups to which the 2-to 8-fold dilutions of the acid decomposition products of agar andagarose and the 2- and 4-fold dilutions of the acid decompositionproduct of bar-shaped agar were added, the absorbance at 590 nm was lessthan one-half of that of the control group to which water was added, andapoptosis corpuscles were observed.

When each of the acid decomposition products was analyzed with normalphase HPLC, agarooligosaccharides such as agarobiose, etc. were detectedfor all the decomposition products.

Example 8

(1) Commercially available agar was suspended in each of the followingaqueous acid solutions at a concentration of 1%.

0.5 M, 1M or 2M citric acid; 0.1 M, 0.5 M, 1 M or 2 M nitric acid; 0.1 Mor 0.5 M sulfuric acid; 0.1 M, 0.5 M or 1 M phosphoric acid; 0.1 Mhydrochloric acid.

The resultant agar suspensions were heated with a microwave oven untilthe agar was dissolved, followed by neutralization with NaOH. Thesolutions were diluted with 2-, 4-, 8-, 16- or 32-folds with distilledwater. Then, an apoptosis-inducing activity and an antiproliferationactivity against HL-60 cells were measured according to the same manneras that described in Example 2-(1).

As a result, agar heated in the above-listed various acids inducedapoptosis in HL-60 cells and inhibited cell proliferation. The resultsare shown in Table 5. The number in Table 5 represents the dilution rateof the dilution added to the group whose absorbance at 590 nm was lessthan one half of that of the control group to which water was added. Inaddition, the number in the parentheses represents the dilution rate ofa solution (prepared, without adding agar, by neutralizing the highestconcentration of the acid used in this Example with NaOH and dilutingthe resulting solution with distilled water) added to the group whoseabsorbance at 590 nm was less than one-half of that of the control groupto which water was added. TABLE 5 0.1 M 0.5 M 1 M 2 M Citric acid  4  8  16(8) Nitric acid 8 16  16(2) Sulfuric acid 8 16(2) Phosphoric acid4  8  16(1) Hydrochloric acid 16

(2) The substances obtained by heating agar in the acids in Example8-(1) were analyzed with normal phase HPLC as follows.

Column: PALPAK type S (4.6×250 mm, manufactured by Takara Shuzo, CA8300)

Solvent A: aqueous 90% acetonitrile solution

Solvent B: aqueous 50% acetonitrile solution

Flow rate: 1 ml/min.

Elution: solvent A (10 min.)>

linear gradient from

solvent A to solvent B (40 min.)>

solvent B (10 min.)

Detection: absorbance at 195 nm

Column temperature: 40° C.

As a result, the samples heated in 0.5 M, 1 M and 2 M citric acid, 0.1M, 0.5 M, 1 M and 2 M nitric acid, 0.1 M and 0.5 M sulfuric acid, 0.1 M,0.5 M and 1 M phosphoric acid, and 0.1 M hydrochloride acid containedagarooligosaccharides such as agarobiose, etc.

The representative result is shown in FIG. 16. That is, FIG. 16illustrates the normal phase HPLC elution pattern of agar heated in 0.5M phosphoric acid. In FIG. 16, the horizontal axis represents theretention time (min.) and the vertical axis represents the absorbance at195 nm.

Example 9

A suspension of 5 g of commercially available agar (Ina agar type S-7,manufactured by Ina Shokuhin Kogyo) in 45 ml of 20, 50 or 100 mM citricacid was heated at 95° C. Samples were obtained after heating for aperiod of time as described below.

For 20 mM citric acid, 310 min., 350 min., 380 min., 440 min. and 530min.

For 50 mM citric acid, 100 min., 120 min., 140 min., 160 min., 180 min.,200 min., 220 min., 240 min., 260 min., 290 min. and 320 min.

For 100 mM citric acid, 60 min., 70 min., 80 min., 90 min., 100 min.,120 min., 140 min., 160 min., 180 min., 200 min., 220 min. and 240 min.

1 μl of 10-fold dilution of each sample was spotted on a silica gel 60sheet F₂₅₄ (manufactured by Merck), developed with1-butanol:ethanol:water=5:5:1 and detected by orcinol-sulfuric acidmethod.

As a result, each sample contained agarooligo-saccharides such asagarobiose, agarotetraose and agarohexaose, etc.

For the samples treated with 20 mM citric acid, the agarooligosaccharidecontent was increased by heating for as long as 350 minutes and,thereafter, remained almost constant.

For the samples treated with 50 mM citric acid, the agarooligosaccharidecontent was increased by heating for as long as 200 minutes and,thereafter, remained almost constant.

For the samples treated with 100 mM citric acid, theagarooligosaccharide content was increased by heating for as long as 160minutes and, thereafter, remained almost constant.

The final agarooligosaccharide content increased with the increase inthe concentration of citric acid.

Each sample was analyzed with the normal phase HPLC according to thesame manner as that described in Example 8-(2). As a result, resultsconsistent with those obtained by thin layer chromatography wereobtained. However, the sample treated with 100 mM citric acid containedmore impurities than that treated with 50 mM citric acid, and theimpurities increased with the increase in the heating time.

Example 10

(1) Agarobiose prepared in Example 3-(1) was dissolved at aconcentration of 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM or 1 mMin water. One microliter of each sample was spotted on a silica gel 60sheet F₂₅₄, developed three times with chloroform:methanol: aceticacid=7:2:2 and color-developed by orcinol-sulfuric acid method. Imagedata of the color-developed sheet was obtained using FOTODYNEFOTO/Analyst Archiver Ecripse (sold by Central Kagaku Bouekisha). Theimage data was image-processed using an image analysis software 1-DBasic (manufactured by Advanced American Biotechnology) and theintensity of the agarobiose spot at each concentration was converted toa numerical value to prepare a calibration curve.

A graph of the calibration curve is shown in FIG. 17. That is, FIG. 17illustrates the calibration curve for agarobiose, and the graph wasprepared by plotting each agarobiose concentration versus the intensityof each spot. In FIG. 17, the horizontal axis represents the agarobioseconcentration (mM) and the vertical axis represents the intensity ofspot. The equation in FIG. 17 represents the relation of the intensityof spot (y) and the agarobiose concentration (x). For a sample whoseagarobiose concentration is unknown, the agarobiose concentration can becalculate from the equation by determining the intensity of spot.

Likewise, calibration curves for agarotetraose, agarohexaose andagarooctaose obtained in Example 5 were prepared.

(2) A suspension of 0.2 g of commercially available agar (Agar Noble) in90 ml of water was heated with a microwave oven and cooled to about roomtemperature. To this resultant solution was added 10 ml of 1 M HCl or 1M citric acid to prepare 0.2% agar solution in 0.1 M HCl or 0.2% agarsolution in 0.1 M citric acid. The solution was heated at 90° C. andsamples were obtained at 5 min., 10 min., 20 min., 30 min., 1 hour, 2hours, 4 hours, 8 hours and 21 hours after initiation of heating. Eachsample was subjected to the thin layer chromatography according to thesame manner as that described in Example 10-(1) and the intensity ofspot was determined to calculate the agarobiose concentration. Eachsample was appropriately diluted to make the agarobiose concentrationwithin a range between 0.05 mM and 1 mM.

In FIGS. 18 and 19, the relation between the heating time and the amountof agarobiose formed in 0.2% agar solution in 0.1 M HCl and 0.2% agarsolution in 0.1 M citric acid. That is, FIG. 18 illustrates the relationbetween the heating time and the amount of agarobiose formed in 0.2%agar solution in 0.1 M HCl. FIG. 19 illustrates the relation between theheating time and the amount of agarobiose formed in 0.2% agar solutionin 0.1 M citric acid. In FIG. 18 and FIG. 19, each horizontal axisrepresents the heating time (hrs.) and the vertical axis represents theagarobiose concentration (mM).

As shown in FIG. 18, in 0.2% agar solution in 0.1 M HCl, the agarobioseconcentration reached the maximum by heating for one hour and reducedthereafter. And, as shown in FIG. 19, in 0.2% agar solution in 0.1 Mcitric acid, the agarobiose concentration increased gradually by heatingas long as 8 hours and reduction was observed at 21 hours. Theagarobiose concentration in a sample obtained by heating 0.2% agarsolution in 0.1 M HCl for 5 minutes, or by heating 0.2% agar solution in0.1 M citric acid for 5, 10, or 20 minutes was below the detectablelimitation.

(3) Agar Noble was suspended in 5, 50 and 500 mM citric acid at aconcentration of 10%, heated at 65, 80 or 95° C. Samples were obtainedat 30 min., 1, 2, 4, 8 or 24 hours after the initiation of heating and,according to the same manner as that described in Example 10-(1), theamount of agarooligosaccharides formed was measured.

As a result, when agar was dissolved in 5 mM citric acid, although smallamounts of agarooligosaccharides were formed at 80° C., they werescarcely formed at 65° C. At 95° C., a large amount of agarobiose wasformed by heating for 8 to 24 hours, and a large amount of agarotetraosewas also formed by heating for 8 to 24 hours. When agar was dissolved in50 mM citric acid, agarooligosaccharides were scarcely formed at 65° C.At 80° C., a large amount of agarobiose was formed by heating for 24hours, and large amounts of agarotetraose, agarohexaose and agarooctaosewere formed by heating for 4 to 8 hours. At 95° C., a large amount ofagarobiose was formed by heating for 24 hours, and large amounts ofagarotetraose, agarohexaose and agarooctaose were formed by heating for4 to 8 hours. When agar was dissolved in 500 mM citric acid, smallamounts of agarooligosaccharides were formed by heating at 65° C. for 4to 24 hours. At 80° C., a large amount of agarobiose was formed byheating for 2 to 24 hours, and large amounts of agarotetraose andagarohexaose were formed by heating for 1 to 6 hours. A large amount ofagarooctaose was formed by heating for 1 to 2 hours. At 95° C., a largeamount of agarobiose was formed by heating for 1 to 24 hours, and alarge amount of agarotetraose was formed by heating for 1 to 2 hours.Large amounts of agarohexaose and agarooctaose were formed by heatingfor 30 minutes to 1 hour.

Examples of hydrolysis by 500 mM citric acid are shown in FIGS. 20 and21. That is, FIG. 20 illustrates agarooligosaccharide formation in 500mM citric acid by heating at 80° C. In FIG. 20, the vertical axisrepresent the amounts of agarooligosaccharides formed (open circle:agarobiose, open triangle: agarotetraose, open square: agarohexaose,symbol x: agarooctaose) and the horizontal axis represents the time.FIG. 21 illustrates the amounts of agarooligosaccharide formation in 500mM citric acid by heating at 95° C. In FIG. 21, the vertical axisrepresents the amounts of agarooligosaccharides formed (open circle:agarobiose, open triangle: agarotetraose, open square: agarohexaose,symbol x: agarooctaose) and the horizontal axis represents the time.

(4) According to the same manner as that described in Example 10-(3),agarooligosaccharide formation in 50, 500 or 1000 mM acetic acid wasmeasured.

As a result, when agar was dissolved in 50 mM acetic acid, small amountsof agarooligosaccharides were formed at 80° C., whileagarooligosaccharides were scarcely formed at 65° C. When agar wasdissolved in 500 mM acetic acid, agarooligosaccharides were scarcelyformed at 65° C. At 80° C. and 95° C., small amounts ofagarooligoshaccharides were formed. When agar was dissolved in 1000 mMacetic acid, agarooligo-shaccharides were scarcely formed at 65° C. At80° C., a large amount of agarobiose was formed by heating for 24 hours,and small amounts of agarotetraose, agarohexaose and agarooctaose wereformed by heating for 8 hours. At 95° C., a large amount of agarobioseformed by heating for 8 hors, and large amounts agarotetraose,agarohexaose and agarooctaose were also formed by heating for 8 hours.

(5) According to the same manner as that described in Example 10-(3),agarooligosaccharide formation in 60, 600 or 1200 mM lactic acid wasmeasured.

As a result, when agar was dissolved in 60 mM lactic acid, small amountsof agarooligosaccharides were formed at 95° C., whileagrooligosaccharides were scarcely formed at 65 and 80° C. When agar wasdissolved in 600 mM lactic acid, large amounts of agarobiose were formedby heating at 80° C. for 8 to 24 hours, and large amounts ofagarotetraose and agarohexaose were formed by heating for 4 to 8 hours.A large amount of agarooctaose was formed by heating for 4 hours. At 95°C., a large amount of agarobiose was formed by heating for 4 to 8 hours,and large amounts of agarotetoraose and agarohexaose were formed byheating for 2 to 6 hours. When agar was dissolved in 1200 mM lacticacid, a large amount of agarobiose was formed by heating at 80° C. for 4to 24 hours, and large amounts of agarotetoraose and agarohexaose wereformed by heating for 2 to 6 hours. A large amount of agarooctose wasformed by heating for 2 hours. At 95° C., a large amount of agarobiosewas formed by heating for 2 to 8 hours, and large amounts ofagarotetraose and agarohexaose were formed by heating for 1 to 2 hours.

Examples of hydrolysis by 1200 mM lactic acid are shown in FIGS. 22 and23. That is, FIG. 22 illustrates agarooligosaccharide formation in 1200mM lactic acid by heating at 80° C. In FIG. 22, the vertical axisrepresents the amounts of agarooligosaccharides formed (open circle:agarobiose, open triangle: agarotetraose, open square: agarohexaose,symbol x: agarooctaose) and the horizontal axis represents the time.FIG. 23 illustrates agarooligosaccharide formation in 1200 mM citricacid by heating at 95° C. In FIG. 23, the vertical axis represents theamounts of agarooligosaccharides formed (open circle: agarobiose, opentriangle: agarotetraose, open square: agarohexaose, symbol x:agarooctaose) and the horizontal axis represents the time.

(6) According to the same manner as that described in Example 10-(3),agarooligosaccharide formation in 20, 200, or 1000 mM malic acid wasmeasured.

As a result, when agar was dissolved in 20 mM malic acid, small amountsof agarooligosaccharides were formed at 95° C., butagarooligosaccharides were scarcely formed at 65° C. and 80° C. Whenagar was dissolved in 200 mM malic acid, small amounts ofagarooligosaccharides were formed by heating at 65° C. for 24 hours. At80° C., a large amount of agarobiose was formed by heating for 8 to 24hours, and a large amount of agarotetoraose was formed by heating for 4to 8 hours. A large amount of agarohexaose was formed by heating for 4hours. A large amount of agarooctaose was formed by heating for 4 hours.At 95° C., a large amount of agarobiose was formed by heating for 4 to 8hours, and a large amount of agarotetraose was formed by heating for 4hours. When agar was dissolved in 1000 mM malic acid, at 65° C., smallamounts of agarooligosaccharides were formed by heating for 24 hours. At80° C., a large amount of agarobiose was formed by heating for 2 to 24hours, and a large amount of agarotetraose was formed by heating for 2to 6 hours. Large amounts of agarohexaose and agarooctaose were formedby heating for 2 hours. At 95° C., a large amount of agarobiose wasformed by heating for 1 to 8 hours, and a large amount of agarotetoraosewas formed by heating for 1 to 2 hours. Large amounts of agarohexaoseand agarooctaose were formed by heating for at 1 hour.

Examples of hydrolysis by 1000 mM malic acid are shown in FIGS. 24 and25. That is, FIG. 24 illustrates agarooligosaccharides formation in 1000mM malic acid by heating at 80° C. In FIG. 24, the vertical axisrepresents the amounts of agarooligosaccharides formed (open circle:agarobiose, open triangle: agarotetraose, open square: agarohexaose,symbol x: agarooctaose) and the horizontal axis represents the time.FIG. 25 illustrates agarooligosaccharide formation in 1000 mM malic acidby heating at 95° C. In FIG. 25, the vertical axis represents theamounts of agarooligosaccharides formed (open circle: agarobiose, opentriangle: agarotetraose, open square: agarohexaose, symbol x:agarooctaose) and the horizontal axis represents time.

(7) Noble agar was suspended in 100, 200, 300, 400, 500, 600, 700, 800,900 or 1000 mM malic acid at a concentration of 10% and the suspensionswere heated at 70, 80 or 90° C. Samples were obtained at 30 min., 1, 2,3, 4, 8, or 24 hours after initiation of heating and, according to thesame manner as that described in Example 10-(3), agaro-oligosaccharideformation was measured.

At the malic acid concentration of 300 mM or more, large amounts ofagarooligosaccharides were formed even by heating at 70° C. for 8 hoursor longer. Examples of hydrolysis in 1000 mM malic acid at 70° C. areshown in FIG. 26. That is, FIG. 26 illustrates agarooligosaccharidesformation in 1000 mM malic acid by heating at 70° C. In FIG. 26, thevertical axis represents the amounts of agarooligosaccharides formed(open circle: agarobiose, open triangle: agarotetraose) and thehorizontal axis represents the time.

Based on the results of Example 10-(3) to (7) as described above,agarooligosaccharides are preferably produced by using an acid such ascitric acid, lactic acid or malic acid at a concentration of several tenmM to several M and heating at 70 to 95° C. for several ten minutes to24 hours.

(8) Agarobiose was determined using F-kit lactose/galactose(manufactured by Boehringer Mannheim, code 176303). In theabove-mentioned method, agarobiose was determined by measuring theconcentration of galactose generated from agarobiose by the action ofS-galactosidase in F-kit.

The determination was carried out according to the instructions attachedto the kit except that β-galactosidase was reacted at 37° C. for 1hours. A calibration curve was prepared using lactose. A molarconcentration (mM) was calculated in terms of lactose, which was thenconverted to agarobiose concentration (mg/ml).

According to the above method, the determination of agarobiose,agarotetraose, agarohexaose and agarooctaose prepared in theabove-mentioned Example was tried. As a result, for agarobiose, thecalculated value agreed with the actually determined value. On the otherhand, agarotetraose, agarohexaose and agarooctaose were notsubstantially detected by the above-mentioned method. Namely, inpractice, it was found that agarooligosaccharides except agarobiose werenot detected by the above-mentioned method and that the agarobioseconcentration in agarooligosaccharides can be measured using theabove-mentioned method.

(9) A mixture of 100 g of commercially available agar (Ina agar typeS-7: manufactured by Ina Shokuhin Kogyo) and 10 g of H-type strongcation exchange resin (Diaion SK104H: manufactured by MitsubishiChemical) was prepared by mixing them in 900 g of desalted water at 95°C. The mixture was stirred at 95° C. for 180 minutes to carry out aciddecomposition of agar. Then, the resulting mixture was cooled to roomtemperature, filtrated by body feed of 1% w/w of activated carbon and0.5% w/w of Celite 545 (manufactured by Celite) to obtain a filtrate.

According to the same manner as that described in Example 8-(2), thefiltrate obtained was analyzed by normal phase HPLC to confirm thatagarobiose, agarotetraose, agarohexaose and agarooctaose were mainlyformed as agrooligosaccharides.

The filtrate was at pH 2.4, and it had the acidity of 1.7, the brix of9.4% and the agarobiose content of 7.4% as measured by using F-kitlactose/galactose described in Example 10-(8).

(10) To 100 g of desalted water was added 18.5 g of H-type strong cationexchange resin (Daiyaion SK104H) and the mixture was stirred at 95° C.At 10 minutes intervals, 10 g of agar (Ina agar type S-7) was added 5times, then 15 g of agar was added 2 times, 20 g of agar was added 3times, and finally 30 g of agar was added. After adding a total of 185 gof agar, the mixture was stirred at 95° C. for 150 minutes and thencooled to room temperature. The resultant mixture was decanted toseparate the resin from the liquid phase. Then, the separated liquidphase was filtrated by body feed of 3% w/w of activated carbon and 0.5%w/w of Celite 545 (manufactured by Celite) to obtain a filtrate.

According to the same manner as that described in Example 8-(2), thefiltrate was analyzed by normal phase HPLC to confirm that agarobiose,agarotetraose, agarohexaose and agarooctaose were mainly formed asagarooligosaccharides.

The filtrate was at pH 1.2 and it had the acidity of 11.9, the brix of64% and the agarobiose content of 24.4% as measured by using F-kitlactose/galactose described in Example 10-(8).

(11) Liquefaction of agar was carried out by preparing a suspensioncontaining 100 g of commercially available agar (Ina agar type S-7) indeionized water having various phosphoric acid concentrations added to avolume of 1 liter, and stirring the resultant suspension at 95° C.

According to the same manner as that described with respect to thesuspension containing phosphoric acid, liquefaction of agar was carriedour by preparing a suspension containing agar in deionized watercontaining 1% w/v citric acid added to a volume of 1 liter. The term“liquefaction” used herein means a state in which gelation does not takeplace even at a freezing point. Time required for achieving such a state(liquefaction time) was measured. In addition, agarobiose contents uponliquefaction and thereafter were measured by using F-kitlactose/galactose described in Example 10-(8). The results are shown inTables 6 and 7. TABLE 6 Phosphate Liquefaction Time held conc. time at95° C. Agarobiose (% w/v) (min.) (min.) (g/l) 0.2 150 150 2.57 180 3.80300 7.97 0.3 120 120 4.88 180 7.07 300 13.90 0.5 110 110 6.09 180 8.48300 21.40 1.0 90 90 7.58 120 18.80

TABLE 7 Citric acid Liquefaction Time held concentration time at 95° C.Agarobiose (% W/V) (min.) (mm.) (g/liter) 1.0 90 90 0.95 120 3.40 1504.09 300 5.70 360 14.80

(1) A mixture of 150 g of commercially available agar (Ina agar typeS-7, manufactured by Ina Shokuhin Kogyo) and 15 g of citric acid(anhydrate) for food additives (manufactured by San-Ei Gen F.F.I. wasmade up to 1.5 liter with deionized water. The mixture was warmed to 92°C. and then held at 92-95° C. for 130 minutes with stirring. Then, themixture was cooled to room temperature and filtrate by body feed of 0.5%of Celite 545 (manufactured by Celite) to obtain a filtrate (agardecomposition oligosaccharide solution). According to the same manner asthat described in Example 8-(2), the filtrate obtained was analyzed bynormal phase HPLC to confirm that agarobiose, agarotetraose,agarohexaose and agarooctaose were mainly formed as saccharidecompounds.

The filtrate was at about pH 2.6 and it had the acidity of 0.92, thebrix of 9.2% and the agarobiose content of 43.1 mM as measured by themethod described in Example 10-(8).

(2) The filtrate (agar decomposition oligosaccharide solution) preparedin Example 11-(1) was diluted 20-folds and to this were added acidulant,sweetener and flavor to prepare soft drinks containing 2.25 mMagarobiose.

The formulations are shown in Tables 8 and 9. Table 8 shows theformulation of a grapefruit soft drink and Table 9 shows the formulationof perilla flavored soft drink.

The components shown in each table were added to water and dissolved toprepare the soft drink, and the soft drink was distributed in 200 mlcans. The soft drink shown in Table 8 was carbonated to prepare acarbonated drink whose gas pressure was 0.8 kg/cm² (20° C.).

The analytical values for each drink are shown in the lower columns ofTables 8 and 9. TABLE 8 Agar decomposition o1igo- 50 ml saccharidesolution 1/7 grapefruit 20 g Vitamin C 0.2 g Citric acid 0.2 g Maltose1.25 g Grapefruit flavor 1 g Desalted water rest Total 1000 ml pH 3.2Acidity* 0.23 Brix 2.2Acidity*: 0.1 N NaOH ml/l0 ml (hereinafter the same)

TABLE 9 Agar decomposition oligo- 50 ml saccharide solution Perillaextract 20 g Vitamin C 0.2 g Citric acid 0.2 g Perilla flavor 0.5 gDesalted water rest Total 1000 ml pH 2.9 Acidity* 0.10 Brix 0.6

Each carbonated drink of the present invention was assessed by 10panelists in a sensory test which scores in five grades (5: good, 1:bad). As a control, a soft drink prepared by using an aqueous citricacid solution having the same acidity instead of the agar decompositionoligosaccharide solution was used.

The average scores obtained in the sensory test for the grapefruittasted ones and those for the perilla flavored ones are shown in Tables10 and 11. TABLE 10 Product of the present invention Control Texturemildness 4.6 3.1 smoothness 4.8 3.0 Flavor balance 4.5 2.9 General 4.63.1 assessment

TABLE 11 Product of the present invention Control Texture mildness 4.52.5 smoothness 4.3 2.4 Flavor balance 4.2 2.5 General 4.3 2.4 assessment

As compared with the control, the products of the present invention wereassessed to have better flavor balance as well as milder and smoothertexture. Thus, the products are drinks having novel tastes. Likewise,the soft drinks without carbonation of the present invention had noveltastes.

(3) Ethyl alcohol was added to each of the soft drinks described inTables 8 and 9 at ethyl alcohol concentrations of 6% v/v or 8% v/v andthe resulting mixtures were distributed in 200 ml cans. The alcoholdrinks were carbonated to prepare the carbonated alcohol drinks of thepresent invention in which gas pressure was 0.8 kg/cm² (20° C.).

As compared with the control which did not contain the agardecomposition oligosaccharide solution, the carbonated alcohol drinks ofthe present invention were to have better flavor balance as well asmilder and smoother texture. Thus, the carbonated alcohol drinks of thepresent invention had novel tastes.

Example 12

(1) A drink containing an agar decomposition product decomposed bycitric acid was prepared as follows. The formulation is shown in Table12. Namely, for Product 1 of the present invention in Table 12, 0.1% w/vof the filtrate prepared in Example 11-(1) (agar oligosaccharidesolution), 0.25% w/v of agar (Ultra Agar AX-30: manufactured by InaShokuhin Kogyo) and 0.07% w/v of citric acid were used. For Product 2 ofthe present invention, 0.25% w/v of agar and 0.08% w/v of citric acidwere used without addition of the agar oligosaccharide solution. Ineither case of Products 1 and 2 of the present invention, the drinkscontaining the agar decomposition products decomposed by citric acidwere prepared by dissolving agar with hot water, mixing it with theother components and heating under acidic conditions at 93° C. for 10seconds, at 93-80° C. for 20 minutes and at 80-75° C. for 15 minutes. Onthe other hand, a control was prepared according to the same formulationas that of Product 2 of the present invention except that heating wascarried out at 93° C. for 10 seconds.

Each drink with thickness containing the agar decomposition productdecomposed by citric acid was assessed by 10 panelists in a sensory testwhich scores in five grades (5: good, 1: bad). The mean scores obtainedin the sensory test are shown in Table 13. TABLE 12 Product 1 Product 2Agar (g) 2.5 2.5 Agar oligosaccharide solution 1.0 0 (ml) 1.5 1.5 1/7grapefruit juice (g) 66.0 66.0 Granulated sugar (g) 0.7 0.8 Citric acid(g) 0.5 0.5 Sodium citrate (g) 2.0 2.0 Flavor (g) rest rest Desaltedwater Total 1000 ml 1000 ml pH 3.68 3.67 Acidity* 1.53 1.57 Brix 7.5 7.4Agarobiose (mM)** 0.06 0.02Agarobiose (mN)** was measured by the method described in Example 10-(8)

TABLE 13 Product Product 1 2 Control Texture mildness 4.4 4.1 3.6smoothness 4.5 4.3 3.8 Flavor balance 4.4 4.2 3.6 General 4.5 4.3 3.7assessment

As compared with the control, Products 1 and 2 of the present inventionwere assessed to have better flavor balance, suitable thickness as wellas milder and smoother texture. Thus, the products were drinks withnovel tastes. The formation of an oligosaccharide for an antioxidant,agarobiose, by heat treatment in the presence of citric acid added inProducts 1 and 2 of the present invention was recognized. Thus, thenovel drinks containing an oligosaccharides for an antioxidant wereprovided.

According to the same manner as that described in Example 10-(8), amounts of agarobiose formed were measured using heating conditions at75° C. for 1 day; at 85° C. for 5 minutes; at 103° C. for 5 minutes; orat 122° C. for 45 seconds in stead of 80-75° C. for 15 minutes. As aresult, it was confirmed that the amounts of agarobiose formed were 0.03mM, 0.02 mM, 0.04 mM and 0.05 mM, respectively, that agarobiose wasformed by heat treatment, and that the more severe the heatingconditions became, the more agarobiose was formed.

(2) Ethyl alcohol was added to Product 1 and Product 2 of the presentinvention, and the control described in Example 12-(1) at ethyl alcoholconcentration of 2% v/v or 4% v/v. The total volume was adjusted byreducing the volume of desalted water which corresponds to that of ethylalcohol added. Thus, alcohol drinks were prepared.

As compared with the control, the alcohol drinks corresponding toProducts 1 and 2 were assessed to have better flavor balance, suitablethickness as well as milder and smoother texture. Thus, the productswere drinks having novel taste.

Additionally, frozen products of the above-described alcohol drinksexhibited good sherbet-like texture.

Example 13

(1) Commercially available agar (Agar Noble) was suspended in 0.1 Nhydrochloric acid at a concentration of 1% and the suspension wastreated at 37° C. for 5 hours, 16 hours or 48 hours. The suspension thustreated was diluted 10-folds with distilled water and analyzed with thinlayer chromatography as described in Example 9. As a result, theformation of small amounts of agarooligosaccharides was observed by thetreatment for 5 hours. The amounts thereof were increased by thetreatment for 16 hours and were further increased by the treatment for48 hours.

(2) Commercially available agar (Agar Noble) was suspended in aphosphate buffered saline or distilled water at a concentration of 1%and the suspension was heated at 121° C. for 4 hours. The suspensionthus heated was diluted 10-folds with distilled water and analyzed withthin layer chromatography as described in Example 9. As a result, theformation of trace amounts of agarooligosaccharides was observed in thesample heat-treated in the phosphate buffered saline. In the sampleheat-treated in distilled water, the formation of clearly more amountsof agarooligosaccharides was observed. The former was at about pH 7after the heat treatment, while the latter was at about pH 5. Aftercooling to room temperature, the former gelated but the latter did not.

Example 14

(1) Agar (Agar Noble) was suspended in 0.1N HCl at a concentration of10% and heated at 100° C. for 19 minutes. TOYOPEARL HW40C (manufacturedby Toso) column (4.4 cm×85 cm) was equilibrated with water and 10 ml ofthe above-mentioned sample was applied to this column. Gel filtrationchromatography was carried out using water as a mobile phase at a flowrate of 1.4 ml/min. The eluted substances were detected using adifferential refractometer and each 7 ml fraction was collected.

Peaks were recognized at elution time 406, 435, 471 and 524 minutes. Theanalysis of the fractions corresponding to respective peaks with thinlayer chromatography as described in Example 9 demonstrated that thesewere agarooctaose, agarohexaose, agarotetraose and agarobiose in thisorder. The fractions were lyophilized to obtain 30 mg agarooctaose, 100mg agarohexaose, 150 mg agarotetraose and 140 mg agarobiose.

(2) Agarobiose and agarohexaose obtained in Example 14-(1) weredissolved in water to prepare 100 mM aqueous solutions thereof. To each25 μl of these solutions was added 50 μl of 100 mM aqueous L-cysteinesolution, followed by addition of 925 μl of phosphate buffered saline.Then, the mixture was treated at 37° C. for 1 hour or 16 hours. The samereaction was repeated except that an aqueous solution containing thesame concentration of L-lysine was used instead of the aqueousL-cysteine solution.

1 μl of a sample from each reaction was spotted on a silica gel sheet 60F₂₅₄, developed with 1-butanol:ethanol:water=5:5:1 and detected byorcinol-sulfate method.

As a result, the spots of agarobiose and agarotetraose were disappearedin the sample from the reaction for 1 hours with L-cysteine. In thesamples from the reaction for 16 hours with L-cysteine and L-lysine, thespots of agarobiose and agarotetraose were disappeared.

When each sample was analyzed with normal phase HPLC as described inExample 8-(2), the results were consistent with those obtained with thethin layer chromatography.

(3) According to the same manner as that described in Example 2-(1), anantiproliferation activity against HL-60 cells was measured by placing10 μl of each sample from the reaction prepared in Example 14-(2) into awell of a 96 well microtiter plate.

As a result, it was observed that the activities were disappeared in thesample whose spots of agarobiose and agarotetraose were disappeared.Namely, antiproliferation activities in the samples from the reaction ofagarobiose and agarotetraose with L-cysteine for 1 hour and from thereaction of agarobiose and agarotetraose with L-cysteine or L-lysine for16 hours were reduced to about 1/10 relative to the same concentrationsof agarobiose and agarotetraose.

Example 15

(1) A suspension of 2.5 g of K-carrageenan (manufactured by Sigma,C-1263) in 50 ml of 0.1 N HCl was heated at 100° C. for 16 minutes. Theresultant solution was cooled to room temperature, neutralized to aboutneutral pH with NaOH, filtrated through Cosmonice filter and separatedwith normal phase HPLC as follows.

-   -   Column: PALPAK type S (4.6×250 mm, manufactured by Takara Shuzo,        CA8300)    -   Solvent A: aqueous 90% acetonitrile solution    -   Solvent B: aqueous 50% acetonitrile solution    -   Flow rate: 1 ml/min.    -   Elution: solvent A (10 minutes)>linear gradient from solvent A        to solvent B (40 minutes)>solvent B (10 minutes)    -   Detection: absorbance at 215 nm    -   Column temperature: 40° C.    -   Amount of sample applied: 50 μl

The separation pattern of normal phase HPLC is shown in FIG. 27. Thatis, FIG. 27 illustrates normal phase HPLC chromatogram of aciddecomposition product of K-carrageenan. The horizontal axis representsthe retention time (min.) and the vertical axis represents theabsorbance at 215 nm.

Each elution peak was fractionated, collected, evaporated to drynessunder reduced pressure and dissolved in 100 μl of water. Each fractionwas sterilized by filtration and, according to the same manner as thatdescribed in Example 2-(1), an antiproliferation activity against HL-60cells was measured. As a result, in groups to which the fractions frompeaks at 27.797, 33.905 to 34.784, and 36.226 to 36.654 min. were added,apoptosis corpuscles were observed. The absorbance at 590 nm thereof waslower than that of the control group to which water was added, and cellproliferation was inhibited.

The fraction from the peak at elution time 27.797 min. was separated 12times under the above-mentioned HPLC conditions and the fractions werecombined and evaporated to dryness under reduced pressure to obtain anapoptosis-inducing and carcinostatic substance.

(2) Mass spectrometry of the apoptosis-inducing and carcinostaticsubstance described in Example 15-(1) was carried out using DX302 massspectrometer (manufactured by Nippon Denshi). Glycerol was used as amatrix and the measurement was performed with negative ion mode.

FAB-MS

m/z 403 [M-H]⁻

-   -   495 [M+glycerol-H]⁻

The results are shown in FIG. 28. That is, FIG. 28 illustrates massspectrum of the apoptosis-inducing and carcinostatic substance. Thehorizontal axis represents the m/z value and the vertical axisrepresents the relative intensity.

A nuclear magnetic resonance spectrum of the apoptosis-inducing andcarcinostatic substance obtained in Example 15-(1) was measured withJNM-A500 nuclear magnetic resonance apparatus (manufactured by NipponDenshi).

In FIG. 29, ¹H-NMR spectrum of the apoptosis-inducing and carcinostaticsubstance is shown. In FIG. 29, the horizontal axis represents thechemical shift value and the vertical axis represents the signalintensity.

Based on these analytical results of mass spectrometry and ¹H-NMR, theapoptosis-inducing and carcinostatic substance described in Example15-(1) was identified as κ-carabiose[β-D-galactopyranosyl-4-sulfate-(1>4)-3,6-anhydro-D-galactose].

In view of the above, it has been found that the apoptosis-inducing andcarcinostatic substance obtained in Example 15-(1) is κ-carabiose.

(3) κ-Carabiose obtained in Example 15-(1) was dissolved in water at aconcentration of 1.56 mM, 10 μl of the solution was placed in a well ofa 96 well microtiter plate and, according to the same manner as thatdescribed in Example 2-(1), an apoptosis-inducing activity and anantiproliferation activity were measured. As a result, apoptosiscorpuscles were observed under an optical microscope and, as comparedwith a control group to which water was added, cell proliferation in thegroup to which κ-carabiose was added was suppressed by about 70%.Therefore, κ-carabiose induced apoptosis in HL-60 cells and inhibitedcell proliferation at 156 μM.

Example 16

(1) A suspension of 4.5 g of commercially available agar powder(manufactured by Wako Pure Chemical Industries, Ltd.) in 150 ml of 0.1 NHCl was heated with a microwave oven. The resultant solution was held ona boiling bath for 10 minutes. After heating, the solution was allowedto cool to room temperature and insoluble materials were removed bycentrifugation. The supernatant was then collected and adjusted to pH6.8 with 1 N sodium hydroxide. To 150 ml of the supernatant was addedthe equal volume of ethyl acetate, and the mixture was stirredvigorously and partitioned between the ethyl acetate phase and theaqueous phase. The partitioned aqueous phase was evaporated to drynesswith an evaporator and the residue was dissolved in 150 ml of wateragain. Insoluble materials were removed by centrifugation to obtain asupernatant. The ethyl acetate phase was evaporated to dryness with anevaporator, dissolved in 100 ml of ion-exchanged water and adjusted topH 6.5 with 1 N sodium hydroxide.

The ethyl acetate phase and the aqueous phase were sterilized byfiltration with a filter of 0.2 μm pore size (manufactured by Corning),diluted 10-, 20- and 30-folds with water and, according to the samemanner as that described in Example 2-(1), an antiproliferation activityagainst HL-60 cells was measured. As a result, an antiproliferationactivity was observed in the aqueous phase, but was not in the ethylacetate phase.

50 ml of the aqueous phase solution thus prepared was subjected to gelfiltration with Cellulofine GCL-25 column (41×905 mm). The eluent was0.2 M NaCl containing 10% ethanol. The elution pattern is shown in FIG.30. That is,

FIG. 30 illustrates the results of gel filtration with CellulofineGCL-25 column. In FIG. 30, the vertical axis represents the saccharidecontent in the eluate measured by phenol-sulfuric acid method(absorbance at 490 nm: closed circle) and the horizontal axis representsthe fraction number (10 ml/fraction).

1 μl of each eluted fraction was spotted on a silica gel sheet 60 F₂₅₄(manufactured by Merck) and developed with 1-butanol:aceticacid:water=4:1:2. Orcinol reagent [prepared by dissolving 400 mg oforcinol monohydrate (manufactured by Wako Pure Chemical Industries,Ltd.) in 22.8 ml of sulfuric acid and adding thereto water to make thetotal volume up to 200 ml] was sprayed and the sheet was heated on a hotplate heated at 150° C. to observe the spots.

Every 5 fractions of Fraction Nos. 40 to 120 whose spots were confirmedby the above-mentioned TLC analysis were combined and sterilized byfiltration. Then, an antiproliferation activity against HL-60 cells wasmeasured. As a result, Fraction Nos. 86 to 90 had the strongestantiproliferation activity.

(2) Fraction Nos. 86 to 88 were recovered, and evaporated to drynesswith an evaporator to obtain 0.94 g of powder. The powder obtained wasdissolved in 30 ml of 90% ethanol, and then white precipitate wasremoved using 5C filter (manufactured by ADVANTEC). The resultant wassubjected to gel filtration with Sephadex LH-20 column (35×650 mm). Theeluent was 90% ethanol. The elution pattern was shown in FIG. 31. Thatis, FIG. 31 illustrates the results of gel filtration with SephadexLH-20 column. In FIG. 31, the vertical axis represents the saccharidecontent in the eluate measured by phenol-sulfuric acid method(absorbance at 490 nm: closed circle) and the horizontal axis representsthe fraction number (10 ml/fraction).

Each eluate fraction was analyzed with a silica gel sheet as describedabove.

Components detected by TLC analysis were roughly divided into fivegroups, i.e., Fraction Nos. 30 to 35, 36 to 40, 41 to 44, 45 to 48 and49 to 53. For each group, 125 μl portions from the respective fractionsof the group were combined and evaporated to dryness with an evaporator.The residue was dissolved in 500 μl of ion-exchanged water and itsantiproliferation activity against HL-60 cells was measured.

Fraction Nos. 36 to 40 which had the strongest antiproliferationactivity against HL-60 cells were evaporated to dryness with anevaporator, and dissolved in 5 ml of 1-butanol:acetic acid:water=4:1:2.The solution was applied to column (20×710 mm) packed with silica gel 60F₂₅₄ and washed with 1-butanol:acetic acid:water=4:1:2. As the eluent,1-butanol:acetic acid:water=4:1:2 was used (3 ml/fraction).

Each eluted fraction was analyzed by using a silica gel sheet asdescribed above. As a result, components detected were roughly dividedinto five groups as follows: Fraction Nos. 46 to 52 (group 1), 60 to 70(group 2), 72 to 84 (group 3), 86 to 94 (group 4) and 96 to 120 (group5).

Each group was evaporated to dryness with an evaporator, dissolved in 5ml of ion-exchanged water and filtrated through a filter having poresize of 0.45 μm (manufactured by IWAKI). An antiproliferation activityagainst HL-60 cells of each solution obtained was measured. As a result,an antiproliferation activity was observed in the groups 3, 4 and 5.

Regarding the structure of the substance contained in the groups 4 and5, it was confirmed to be agarobiose by TLC analysis and massspectrometry.

As for the structure of the substance contained in the group 3, it wasconfirmed to beS-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose by massspectrometry and NMR analysis. FIG. 32 illustrates the mass spectrum ofβ-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose. Thehorizontal axis represents the m/z value and the vertical axisrepresents the relative intensity (%). And, FIG. 33 illustrates the¹H-NMR spectrum of beβ-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose. Thehorizontal axis represents the chemical shift value (ppm) and thevertical axis represents the signal intensity (%).

It was found that, like agarobiose,S-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose had astrong antiproliferation activity against HL-60 cells.

Example 17

(1) Inhibitory activity of the saccharides derived from agar obtained inExample 3 against lipid peroxide radical production was measured asfollows.

Staphylococcus aureus 3A (National Collection Of Type Culture, NCTC8319) was inoculated into 5 ml of brain heart infusion medium(manufactured by Difco, 0037-17-8) and cultured at 37° C. overnight. Thebacterial cells were collected by centrifugation, washed with phosphatebuffered saline 3 times and then suspended in phosphate buffered salineat a concentration of 1×10⁷ colony forming units/ml. A mixture of 100 μlof the cell suspension, 100 μl of an aqueous sample solution, 100 μl ofaqueous 1 mg/ml methemoglobin (manufactured by Sigma M9250) solution,600 μl of phosphated buffered saline and 100 μl of aqueous 50 mMtert-butyl hydroperoxide (manufactured by Katayama Kagaku, 03-4990)solution were reacted at 37° C. for 30 minutes. To the reaction mixturewas added 1 ml of 2×NMP medium [prepared by dissolving 8 g of nutrientbroth (manufactured by Difco, 0003-01-6), 5 g of trypton (manufacturedby Difco, 0123-17-3), 5 g of NaCl, 10 g of mannitol (manufactured byNacalai Tesque, 213-03) and 0.035 g of phenol red (manufactured byNacalai Tesque, 268-07) in distilled water to make the volume up to 500ml. The pH was adjusted to 7.5 with NaOH and then the mixture wassterilized by filtration] to stop the reaction. The resultant mixturewas diluted every 3-folds with NMP medium (prepared by diluting 2×NMPmedium 2-folds with sterilized water) to prepare 12 serial dilutions and160 μl of each dilution was placed in each well of a 96-well microtiterplate. The plate was incubated at 37° C. overnight. Color of the mediumwas observed with the naked eye and the sample contained in a well inwhich the color of the medium changed from red to yellow by growth ofthe bacterium was identified as that having an activity of inhibitinglipid peroxide radical production.

The results are shown in Table 14. In Table 14, + represents the samplein which the growth of the bacterium was observed, and—represents thesample in which the growth of the bacterium was not observed. Theconcentration shown in the uppermost line of the table is that of thesample in the reaction mixture in which the sample was reacted withtert-butyl hydroperoxide and the bacterial cells at 37° C. for 30minutes. TABLE 14 0.1 mM 1 mM Agarobiose + + Agarotetraose − + Galactose− −

As seen from the above results, a strong activity of inhibiting lipidperoxide radical production was found in agarobiose and agarotetraose.Similar activity was confirmed in carabiose and3,6-anhydro-2-O-methyl-L-galactose.

(2) A suspension of 5 g of commercially available agar (Ina agar typeS-7, manufactured by Ina Shokuhin Kogyo) in 45 ml of 50 mM citric acidwas heated at 93° C. for 155 minutes and adjusted to pH 6 with NaOH toprepare a sample (citric acid treated sample). Likewise, a suspension ofthe same agar in 45 ml of 100 mM hydrochloric acid was heated at 95° C.for 13 minutes and adjusted to pH 6 with NaOH to prepare a sample(hydrochloric acid treated sample). Both samples were diluted with waterto give 1-, 2-, 4-, 8-, 10- and 100-fold dilutions and, according to thesame manner as that described in Example 17-(1), an activity ofinhibiting lipid peroxide radical production inhibitory activity thereofwas determined. As a result, in both of the citric acid treated sampleand the hydrochloric acid treated sample, the activity was confirmed upto 10-fold dilutions and both had equivalent activities of inhibitinglipid peroxide radical production.

Example 18

Inhibitory activity of agarobiose against lymphocyte blastgenesisinduced by Concanavalin A (Con A)

A spleen was taken out from a ddY mouse (Nippon SLC; male, 7 weeks old),finely minced and suspended in RPMI-1640 medium (Gibco) containing 10%fetal bovine serum (HyClone) to obtain a single cell suspension. Thecell suspension was seeded into a plastic Petri dish, incubated at 37°C. for 2 hours in a carbon dioxide incubator. Adhesive cells adhered tothe Petri dish were removed and non-adhesive cells were used as spleenlymphocytes. 200 μl of 2×10⁶ cells/ml spleen lymphocytes suspension wasseeded into each well of 96 well microtiter plate. Agarobiose at varyingconcentration was added to the wells other than the control well.Furthermore, to all the wells was added 5 μg of Con A (Nacalai Tesque)and the plate was incubated at 37° C. for one day in a carbon dioxideincubator. After incubation, 1 μCi of ³H-thymidine was added to eachwell and incubation was continued for additional one day. Then, itsuptake into cells was measured using a liquid scintillation counter.

The results are shown in FIG. 34. FIG. 34 illustrates the relationbetween the agarobiose concentration and the ³H-thymidine uptake inlymphocyte blastgenesis induced by Con A. The horizontal axis representsthe agarobiose concentration and the vertical axis represents the³H-thymidine uptake (cpm). The open bar and the shaded bar represent the³H-thymidine uptake without stimulation and with stimulation by Con A,respectively. As seen from FIG. 34, agarobiose exhibits thedose-dependent inhibitory activity against mouse lymphocyteproliferation stimulated by mitogen, and almost completely inhibits theproliferation at 100 μg/ml. Thus, the inhibitory activity of agarobioseagainst lymphocyte activation has been recognized. For3,6-anhydrogalacto-pyranose, agarotetraose, agarohexaose, agarooctaose,carabiose and 3,6-anhydro-2-O-methyl-L-galactose, similar activitieshave also been recognized.

Example 19

Inhibitory activity of agarobiose against mixed lymphocyte reaction.

Spleens were taken out from a BALB/c mouse (Nippon SLC; male, 6 weeksold) and a C57BL/6 mouse (Nippon SLC; male, 6 weeks old) and spleenlymphocytes were obtained by the above-described method. Each cellsuspension was adjusted to a concentration of 2×10⁶ cell/ml, 100 μlportions from respective suspensions were mixed together and seeded in a96 well microtiter plate. Agarobiose at varying concentration was addedto the wells other than the control well, and the plate was incubated at37° C. for 4 days in a carbon dioxide incubator. After incubation, 1 μCiof ³H-thymidine was added to each well, and the plate was incubated foradditional 1 day. Its uptake into cells was measured using a liquidscintillation counter.

The results are shown in FIG. 35. That is, FIG. 35 illustrates therelation between the agarobiose concentration and the ³H-thymidineuptake in the mixed lymphocyte reaction. The horizontal axis representsthe agarobiose concentration and the vertical axis represents³H-thymidine uptake (cpm). The open bar and the shaded bar represent³H-thymidine uptake in case where cells from either one of the lineswere used independently, and in case where mixed cells from both of thelines were used, respectively. As is seen from FIG. 35, agarobiose hasthe dose-dependent inhibitory activity against lymphocytes activation bystimulation with an alloantigen, and almost completely inhibits thelymphocytes activation at 10 μg/ml. Thus, the inhibitory activityagainst lymphocyte activation of agarobiose has been recognized. For3,6-anhydrogalactopyranose, agarotetraose, agarohexaose, agarooctaose,carabiose and 3,6-anhydro-2-O-methyl-L-galactose, similar activitieshave also been recognized.

Example 20

(1) RAW 264.7 cells (ATCC TIB 71) were suspended in phenol red-freeDulbecco's modified Eagle's medium containing 10% fetal bovine serum(manufactured by Gibco) and 2 mM L-glutamine (manufactured by LifeTechnologies Oriental, 25030-149) at a concentration of 3×10⁵ cells/ml,and 500 μl portions thereof were seeded to respective wells of a 48-wellmicrotiter plate and incubated at 37° C. for 12 hours in the presence of5% CO₂. To each well were added 10 μl of 25 μg/ml lipopolysaccharide(LPS, manufactured by Sigma, L-2012) and 10 μl of aqueous 5000, 1500,500, 150 or 50 μM agarobiose or neoagarobiose (manufactured by Sigma,G4410) solution, and the plate was incubated for additional 12 hours.Then, concentration of NO₂ ⁻ produced by oxidation of NO in the mediumwas measured. As control groups, a group to which LPS was not added anda group to which agarobiose or neoagarobiose was not added wereprovided.

After incubating as described above, 100 μl of 4% Greece reagent(manufactured by Sigma, G4410) was added to 100 μl of the medium, andthe mixture was allowed to stand for 15 minutes at room temperature.Then, the absorbance at 490 nm was measured. NO₂ ⁻ concentration in themedium was calculated with reference to a calibration curve prepared byusing NaNO₂ at given concentrations dissolved in the same medium as thatdescribed above. All the measurements were carried out in triplicate.

As a result, agarobiose dose-dependently inhibited NO production inducedby LPS, while neoagarobiose did not. The results are shown in FIGS. 36and 37. That is, FIG. 36 illustrates the NO₂ ⁻ concentration in themedium incubated under the respective incubation conditions withaddition of agarobiose. FIG. 37 illustrates the NO₂ ⁻ concentration inthe medium incubated under respective incubation conditions withaddition of neoagarobiose. In FIGS. 36 and 37, the horizontal axes therepresent incubation conditions and the vertical axes represent the NO₂⁻ concentration (μM)

When 3,6-anhydrogalactopyranose, carabiose, agarotetraose, agarohexaose,agarooctaose and 3,6-anhydro-2-O-methyl-L-galactose were used instead ofagarobiose, the similar results were obtained.

(2) A suspension of 5 g of commercially available agar (Ina agar typeS-7, manufactured by Ina Shokuhin Kogyo) in 45 ml of 0.1 N HCl wastreated at 95° C. for 13 minutes. After cooling to room temperature, thesuspension was neutralized with NaOH and filtered through 0.22 μmMILLEX-GP filter (manufactured by Milipore, SLGPR25LS). For this sample(agar decomposition product with hydrochloric acid) and agardecomposition oligosaccharide solution as described in Example 11-(1)(agar decomposition product with citric acid), according to the samemanner as that described in Example 20-(1), an activity of inhibiting NOproduction was measured. Namely, 10 μl of 25 μg/ml LPS and 10 μl of a20-fold dilution of the sample mentioned above were added to wells of a48 well microtiter plate containing RAW264.7 cells which had beenincubated in the wells. The measurement was carried out with the culturemedium. As control groups, a group to which LPS was not added, a groupto which a sample was not added and a group to which 2.5 mM citric acidwas added were provided. All the measurement were carried out induplicate.

As a result, both of the agar decomposition product with hydrochloricacid and the agar decomposition product with citric acid inhibited theNO production induced by LPS. The results are shown in FIG. 38. That is,FIG. 38 illustrates the NO₂ ⁻ concentration in the medium cultured withaddition of the agar decomposition product with hydrochloric acid oragar decomposition product with citric acid. In FIG. 38, the horizontalaxis represents the incubation conditions and the vertical axisrepresents the NO₂ ⁻ concentration (μM).

(3) According to the same manner as that described in Example 20-(2), aninhibitory activity against NO production was evaluated by using anaqueous 100 mM galactose (manufactured by Nacalai Tesque, code 165-11)or 100 mM 3,6-anhydro-D-galactose (manufactured by Funakoshi, codeG0002) solution.

As a result, 3,6-anhydro-D-galactose inhibited NO production, whilegalactose did not. The results are shown in FIG. 39. That is, FIG. 39illustrates the NO₂ ⁻ concentration in the medium cultured with additionof 3,6-anhydro-D-galactose or galactose. In FIG. 39, the horizontal axisrepresents the incubation conditions and the vertical axis representsthe NO₂ ⁻ concentration (μM).

(4) RAW 264.7 cells were suspended in the Dulbecco's modified Eagle'smedium described in Example 20-(1) at a concentration of 3×10⁵ cells/ml,and 500 μl portions thereof were placed in respective wells of a 48 wellmicrotiter plate. The plate was incubated for 37° C. for 10 hours in thepresence of 5% carbon dioxide. To the wells was added 10 μl of aqueous5,000 μM agarobiose solution and incubated for additional 1, 2, 4 or 6hours. Then, the culture supernatant was removed from the well and toeach well were added 500 μl of fresh Dulbecco's modified Eagle's mediumand then 10 μl of aqueous 2.5 μg/ml LPS and aqueous 800 U/mlinterferon-γ (IFN-γ, sold by Cosmobio, GZM-MG-IFN) solution. The platewas incubated for 1 hour. Then, the culture supernatant was removed fromthe well and to each well was added 500 μl of fresh Dulbecco's modifiedEagle's medium and the plate was incubated for additional 16 hours. Theconcentration of NO₂ ⁻-produced by oxidation of NO in the medium wasmeasured according to the same manner as that described in Example20-(1). As control groups, a group to which neither LPS nor IFN-γ wasadded and a group to which agarobiose was not added were provided. Allthe measurements were carried out in duplicate.

As a result, the longer the pre-incubation time was, the higher theinhibition of NO production by agarobiose was. Namely, by addition ofagarobiose to a cell culture medium beforehand, NO production induced byLPS and IFN-γ could be inhibited and prevented. The results are shown inFIG. 40. FIG. 40 illustrates the NO₂ ⁻ concentration in the mediumcultured under respective incubation conditions. In FIG. 40, thehorizontal axis represents the incubation conditions and the verticalaxis represents the NO₂ ⁻ concentration. For 3,6-anhydrogalactopyranose,agarotetraose, agarohexaose, agarooctaose, carabiose and3,6-anhydro-2-O-methyl-L-galactose, the similar activities have alsobeen recognized.

Example 21

(1) Agar powder (manufactured by Wako Pure Chemical Industries, Ltd.)was added to 50 mM citric acid solution at a final concentration of 3%.The resultant was heat-treated at 95° C. for 160 minutes to prepare anoligosaccharide solution for a carcinostatic test.

Male nude mice (SPF/VAFBalb/cAnNCrj-nu, 4 weeks old) were purchased fromNippon Charles River and pre-bred for 1 week. Human colon cancer cellline HCT116 (ATCC CCL-247) were transplanted subcutaneously to the miceat 1.5×10⁶ cells/mouse.

After 2 weeks from the transplantation of the colon cancer cell line,the above oligosaccharide solution for the carcinostatic test which wasadjusted to pH 6.5 just before use was freely given to the mice asdrinking water for 5 days per week. The average of daily intake per onemouse was 3.5 ml. Furthermore, MF manufactured by Oriental Yeast wasfreely given to the mice as feed.

After 4 weeks from the beginning of administration of oligosaccharides,the solid cancer was removed from each mouse that receivedoligosaccharides and the weight of each solid cancer was compared withthat of a control to which normal water was given. This test was carriedout using 10 mice per one group.

As a result, a significant activity of inhibiting cancer cell growth wasobserved in the group to which the sample for the carcinostatic test wasadministrated orally, and a strong carcinostatic activity was observedin the group to which the oligosaccharides derived from agar wasadministrated orally.

The results are shown in FIG. 41. That is, FIG. 41 illustrates thecarcinostatic activity of the oligosaccharides of the present invention.The vertical axis represents the weight of solid cancer (g) and thehorizontal axis represents the control group and the group administratedwith oligosaccharide.

In one mouse of the group to which the neutralized sample for thecarcinostatic test was administrated orally, the cancer was completelydisappeared.

(2) A carcinostatic test was carried out against Ehrlich's ascitescarcinoma using the agar decomposition oligosaccharide solution asdescribed in Example 11-(1).

Ehrlich's carcinoma cells were injected to female ddY line mice (5 weeksold, weighing about 25 g) intraperitoneally (1.2×10⁶ cells/mouse) andaverage days of survival and prolongation rates were calculated based onthe number of survived animals.

Mice were divided into 3 group each consisting of 8 mice. One was acontrol, and other two groups received 3.3-fold dilution and 16.7-folddilution of the agar decomposition oligosaccharide solution described inExample 11-(1), respectively. Namely, each aqueous dilution of the agardecomposition oligosaccharide solution prepared in Example 11-(1) wasprepared and was freely given to the mice from 3 days before cancer celladministration. For the group to which the 3.3-fold dilution of the agardecomposition oligosaccharide solution was given, the daily intake ofthe dilution was 5 ml/day/mouse. For the group to which the 16.7-folddilution of the agar decomposition oligosaccharide solution was given,the daily intake of the dilution was 6 ml/day/mouse. And, for thecontrol group, the daily intake of water was 7 ml/day.

As a result, while the average days of survival of the control group was11.8 days for the control group, the average days of survival for thegroups received the 3.3-fold dilution and the 16.7-fold dilution were19.8 days and 14.4 days, and the prolongation rates were 168% and 122%,respectively. Thus, a significant prolongation effect was recognized.

Example 22

To Wistar line rat (male, 5 weeks old, weighing about 150 g; Nippon SLC)were injected 100 μg of ovalbumin (OA; Sigma) and 1 ml of alum (tradename: Imject Alum; Piace) intraperitoneally to sensitized the rat. After14 days, peripheral blood was collected from the abdominal aorta of therat and the serum was used as anti-OA antibody.

The back part of Wistar line rat (male, 7 weeks old, weighing about 200g; Nippon SLC) was shaved and 100 μl of the anti-OA antibody wasinjected subcutaneously to that part to give passive sensitization.Forty-eight hours after sensitization, 2 ml of agar decompositionoligosaccharide solution described in Example 11-(1) or its 10-folddilution was administrated intraperitoneally to 4 rats of each group. Torats of a control group, 2 ml of water was administratedintraperitoneally.

Thirty minutes after administration, PCA was raised by injection of 1 mlof saline containing 0.1% OA and 0.5% Evan's blue (Nacalai Tesque) tothe tail vein. Thirty minutes after the induction with antigen, ratswere killed by decapitating and bleeding, and the skin of the back sitewhere the pigment was leaked was removed and collected.

The collected skins were soaked in 1 ml of 1 N KCl (Nacalai Tesque) andallowed to stand overnight. Then, the pigment was extracted by adding 9ml of acetone solution (Nacalai Tesque) containing 0.6 N H₃PO₄ (Merck)and the absorbance at 620 nm was measured using an ELISA reader. Theamount of the pigment leaked from the skin was calculated from acalibration curve of Evan's blue.

The results are shown in FIG. 42. That is, FIG. 42 illustratesinhibition of PCA by the oligosaccharides of the present invention. InFIG. 42, the vertical axis represents the amount of leaked pigment(μg/site), and the horizontal axis represents the agar decompositionoligosaccharide solution used.

As shown in FIG. 42, one half or more pigment leakage by PCA wasinhibited by administration of the agar decomposition oligosaccharidesolution and, as compared with the control, significant difference(p<0.05) was exhibited.

For 3,6-anhydrogalactopyranose, agarobiose, agarotetraose, agarohexaose,agarooctaose, carabiose and 3,6-anhydro-2-O-methyl-L-galactose, thesimilar activities have also been recognized.

Example 23

Mouse melanoma cell B16BL6 suspended in RPMI-1640 containing 10% FBS wasplaced in a 6 well plate at a concentration of 5×10⁴ cells/2 mlmedium/well and incubated at 37° C. On the 2nd day, 100 μl of agarobiosesolution (2 mg/ml to 0.2 mg/ml) was added thereto, and on the 7th day,the medium was changed and, at the same time, 100 μl of agarobiosesolution (2 mg/ml to 0.2 mg/ml) was added thereto. On the 8th day, thecells were collected, DNA, RNA and protein were decomposed, and then theabsorbance at 400 nm was measured to examine an activity of inhibitingmelanin production.

Namely, after removing the medium by suction, 0.3 ml of 0.25% trypsindissolved in 20 mM EDTA solution was added to each well and the platewas incubated at 37° C. for 10 minutes. Then, 2 ml of the fresh mediumwas added to the well and the cells were suspended. The suspension wascollected into a test tube. The medium was then removed bycentrifugation and the cells were suspended in 2 ml of PBS andcentrifuged again. After removing the supernatant, 30 μl of 50 mM sodiumacetate buffer (pH 5.0) containing 5 mM manganese chloride and 1 μl ofU/ml DNase I (manufactured by Takara Shuzo) were added to the cells andthoroughly mixed. The mixture was incubated at 37° C. for 2 hours todecompose DNA. Then, 1 μl of 10 mg/ml ribonuclease A (manufactured bySigma) was added to the mixture and the resultant mixture was incubatedat 50° C. for 1 hour to decompose RNA. Finally, 100 mM Tris-hydrochloricacid buffer (pH 7.8) containing 100 μg/ml proteinase K (manufactured bySigma), 0.1% Triton x and 10 mM EDTA was added thereto to make the totalvolume up to 200 μl for 2×10⁶ cells, and the mixture was incubated at37° C. for 16 hours and then the absorbance at 400 nm was measured.

The result was shown in Table 15. As shown in Table 15, activity of theinhibiting melanin production was recognized at agarobioseconcentrations of 50 and 100 μg/ml and the beautifying/whitening effectof agarobiose was recognized. For agarotetraose, agarohexaose,agarooctaose, carabiose and 3,6-anhydro-2-O-methyl-L-galactose, thesimilar activities have also been recognized. TABLE 15 AgarobioseAbsorbance at 400 nm μg/ml mean ± SD 100 0.383 ± 0.007  50 0.392 ± 0.172 10 0.521 ± 0.256 control 0.487 ± 0.038Note: The measurement was carried out in triplicate; 100 μl of themedium was added to the control.

As described above, according to the present invention, there isprovided the functional substances which are useful as activeingredients for compositions for inducing apoptosis, carcinostaticcompositions, antioxidants such as inhibitors of active oxygenproduction, inhibitors of lipid peroxide radical production andinhibitors of NO production, and immunoregulators, and which are themembers selected from the group consisting of the compounds selectedfrom the group consisting of 3,6-anhydrogalactopyranose, a aldehyde anda hydrate thereof, and 2-O-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate, and thesoluble saccharides containing said compounds, for example, agarobiose,agarotetraose, agarohexaose, agarooctaose, carabiose,3,6-anhydro-2-O-methyl-L-galactose, etc. produced by acid decompositionunder acidic condition below pH 7 and/or enzymatic digestion ofsubstances containing the above-mentioned compounds.

These substances are useful as active ingredients of pharmaceuticalcompositions such as compositions for inducing apoptosis, carcinostaticcompositions, antioxidants for medical use such as inhibitors of activeoxygen production, inhibitors of NO production, etc., immunoregulators,and anti-allergic agents. And, the foods or drinks comprising, producedby adding thereto and/or diluting saccharides selected from thesesaccharides are useful for functional foods or drinks having an activitysuch as an activity of inducing apoptosis, a carcinostatic activity, anantioxidant activity such as an activity of inhibiting active oxygenproduction, an activity of inhibiting NO production, an immunoregulatoryactivity and an anti-allergic activity. Thus, there is provided foods ordrinks which induce apoptosis in cells in lesions in patients sufferedfrom cancers or viral diseases and, therefor, are effective inpreventing or ameliorating the disease states of these diseases. In acase of a cancer of a digestive organ such as colon cancer and stomachcancer, among others, since apoptosis can be induced in tumor cells uponoral intake of the above-mentioned compounds of the present invention infoods or drinks, the foods or drinks of the present invention haveexcellent effects on the prevention or amelioration of the disease stateof a cancer of a digestive organ. Furthermore, the above-mentioned foodsor drinks are useful foods or drinks for opposing oxidative stress onthe basis of their antioxidant activities such as the activity ofinhibiting the active oxygen production.

In addition, the functional substances of the present invention are alsouseful as saccharides for an antioxidant for inhibition of active oxygenproduction, and the foods or drinks comprising, produced by addingthereto and/or produced by diluting the saccharides for an antioxidantof the present invention are useful as those for ameliorating thedisease states of diseases caused by oxidizing substances in a livingbody such as active oxygen. Furthermore, the foods or drinks of thepresent invention are effective for amelioration or prevention ofconstipation by the activity of their active ingredients, i.e., a memberselected from the group consisting of the compound selected from thegroup consisting of 3,6-anhydrogalactopyranose, an aldehyde, and2-O-methylated derivatives thereof and/or the saccharide containing saidcompound.

The saccharides for an antioxidant provided by the present invention areuseful as novel functional saccharides which provide antioxidantactivities such as an activity of inhibiting active oxygen production tofoods or drinks.

The functional substances of the present invention have a freshnesskeeping activity and are very useful for keeping taste or freshness offoods or perishables.

Furthermore, the cosmetic compositions comprising the saccharides of thepresent invention are useful as those for beautifying/whitening ormoisturizing.

According to the present invention, there is also provided acidic foodsor drinks comprising, produced by adding thereto and/or produced bydiluting the functional substances. In the production of such foods ordrinks, factors which influence to the contents of the functionalsubstances have been substantially eliminated and, therefore, veryuseful foods or drinks having high contains of the functional substancesare obtained. Moreover, the acidulant prepared in the presence of anorganic acid is also useful as a novel acidulant having good taste andfunctions.

1. A method for treating a disease that requires induction of apoptosisfor its treatment, a carcinomatous disease, a disease that requiresprotection against oxidation for its treatment, a disease that requiresinhibition of active oxygen production for its treatment, a disease thatrequires inhibition of nitric monoxide production for its treatment, adisease that requires inhibition of lipid peroxide radical productionfor its treatment or a disease that requires immunoregulation for itstreatment, the method comprising administering a pharmaceuticalcomposition which comprises as an active ingredient at least one memberselected from the group consisting of: (1) a compound selected from thegroup consisting of 3,6-anhydrogalactopyranose represented by theformula (I):

an aldehyde thereof, a hydrate thereof, and a 2-O-methylated derivativeof said 3,6-anhydrogalactopyranose, said aldehyde or said hydrate; and(2) a soluble saccharide containing the compound at its reducing end. 2.A method according to claim 1, wherein the saccharide is a productproduced by acid decomposition under acidic conditions below pH 7 and/orenzymatic digestion of a substance containing at least one compoundselected from the group consisting of 3,6-anhydrogalactopyranoserepresented by said formula I, an aldehyde or a hydrate thereof, and a2-O-methylated derivative of the 3,6-anhydrogalactopyranose, saidaldehyde or said hydrate.
 3. A method according to claim 2, wherein thesubstance containing at least one compound selected from the groupconsisting of 3,6-anhydrogalactopyranose represented by formula I, analdehyde and a hydrate thereof, and 2-0-methylated derivatives of the3,6-anhydrogalactopyranose, the aldehyde and the hydrate is at least onesubstance selected from the group consisting of agar, agarose andcarrageenan.
 4. A method according to claim 1, wherein the saccharide isat least one saccharide selected from the group consisting ofagarobiose, agarotetraose, agarohexaose, agarooctaose, κ-carabiose, andβ-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose.